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Center for Plant Cell Biology



REU 2003


REU Students and their Summer 2003 Research Programs

Please click on the following student links to see photos and read about their Summer 2003 research programs in CEPCEB laboratories.

James (Jim) Buchino
Kim Carpenter
Marissa Faeidan
Fredrick (Fred) Larabee
Thomas Laughrey
Hsiang-I (Jenny) Lee
Linda Morales
Catrina Romero
Luis Torner
Jacob Vasquez
Rebecca (Beki) Martin

JAMES (JIM) BUCHINO
Transylvania University, KY
My goal in Dr. Walling's Lab is to utilize a combinatorial chemical library to find inhibitors and activators of the leucine amino peptidase (LAP) of the model plant Arabidopsis. This project involves a "Chemical Genetics" approach: the use of small molecules to disrupt proteins to obtain a phenotype, which has similarities to classical genetic methods. A "forward chemical genetics approach" would be to use chemicals to cause a phenotypic change in the organism, allowing a protein target to be determined. I will be using a "reverse chemical genetics approach" to find molecules that influence the protein, which can disrupt or enhance the function of a specific protein within an organism or in an in vitro assay. This method can be advantageous over classical genetics because it is reversible, can be applied at any point during development, and can be used at varying concentrations to modulate a phenotype, to avoid classical lethal mutations.

LAP cleaves the amino acid at the terminal end of a polypeptide. Its production seems to be connected to response to water deficit and wound plants. To study this protein I will perform in vitro enzyme assays with substrate leucine 7-amino-4-methylcoumarin, produces a fluorescent product that is measured to quantify LAP activity. It is anticipated that some of the 10,000 molecules in CEPCEB's chemical genetics library will inhibit or increase the activity of LAP. This has been an excellent summer experience and helped me to make decisions concerning graduate school. I want to thank Dr. Bray, Dr. Bailey-Serres, Dr. Walling, and everyone else involved for giving me such a great opportunity.

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KIM CARPENTER
Western Washington Univ., Bellingham, WA
This summer in Dr. Bailey-Serres' Lab I will be utilizing four genetic stocks of Arabidopsis thaliana in order to better understand the second messengers that may be involved in the induction of alcohol dehydrogenase (ADH) gene expression. I will be working with wild-type Columbia, and the transgenic lines, ADH:GUS, ROPGAP4::DsGUS, DR5:GUS, DR5:GUS: 35S:CA-rop2. The ADH:GUS line contains the Arabidopsis ADH promoter and 5' untranslated region linked to the open reading frame of beta-glucuronidase (GUS), a reporter gene. The ROPGAP4::DsGUS line possesses a transposon insertion into the RopGAP4 gene, a negative regulator of ADH gene expression. DR5 is a synthetic auxin-responsive promoter. This promoter is linked to GUS is in the Columbia background (DR5:GUS) and in a line that expresses a constitutive-active form of the GTPase Rop2 (DR5:GUS; 35S:CA-rop2).

Signal transduction through Rop2 is required for the induction of ADH mRNA accumulation in response to oxygen deprivation. In the ROPGAP4::DsGUS line the signaling pathway cannot be effectively turned off once it is activated. Rop2 is also important in the auxin-mediated formation of lateral roots. Lateral roots form in response to oxygen deprivation, presumably through an auxin-dependent pathway. Changes in hydrogen peroxide are important in the low oxygen induction of ADH expression. It has also been shown by other researchers that auxin can result in the induction of ADH specific activity. I will use these lines to determine if, oxygen deprivation for 0, 6, 12 and 24 hours stimulates the expression of DR5:GUS in lateral root meristems, and if this is altered in any way in the CA:Rop2 background. I will also determine if auxin, 2,4-dichlorophenoxyacetic acid, treatment (10, 20, 30 and 40 µg/ml for 3 hours) stimulates the expression of ADH:GUS or ROPGAP4::DsGUS. Our lab has shown that hydrogen peroxide, generated by treatment with glucose and glucose oxidase (G/GO), stimulates ADH induction in a Rop-dependent manner. Thus, I will determine if G/GO treatment, (0, 0.8, 1.6, 2.4 units/ml for 3 h), stimulates GUS production in the ADH:GUS and DR5:GUS lines, and if this is altered in any way in the CA:Rop2 background. Finally, there is evidence of cross-talk between signaling mediated by hydrogen peroxide and another second messenger, nitric oxide (NO). I will also test whether NO affects reporter gene activity in these lines. To do so, I will treat seedlings with a NO-generator, S-nitroso-N-acetyl-D, L-penicillamine, (SNAP), and with a NO-scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1oxyl-3-oxide potassium salt, (cPTIO) (2 mM for 0, 3, 6, 9 and 24 h). In summary, my goal for this summer is to look at the relationship between auxin, hydrogen peroxide, nitric oxide and Rop signaling pathway in plant responses to oxygen deprivation.

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MARISSA FAEIDAN
University of California, Riverside
I am working for Shou-Wei Ding's lab on a project about programmed cell death and viral suppression of RNA silencing on the experimental plant organism, N. benthamiana. RNA silencing is an extraordinary phenomenon that has rather recently become a resource for studying a vast array of viral diseases in both the plant and animal kingdoms. RNA silencing is used as a host defense mechanism and is thought to be one of the first and foremost steps in determining whether or not RNA or DNA will be eventually expressed. Suppression of gene silencing could be one major way by which plants and even animals are conquered by certain viruses, including HIV. If gene-silencing is suppressed by a viral suppressor protein the outcome is expression of the viral genes and as a result, rapid necrosis.

My project deals with determining if the occurrence of cell death is absolutely necessary in the presence of a gene-silencing suppressor, the 2b protein of cucumber mosaic virus (CMV). When GFP105, a gene that encodes proteins that allow green fluorescence to illuminate, is over expressed by infiltration into a GFP transgenic plant, gene silencing is triggered under normal conditions. Degradation of GFP RNA is thought to occur as indicated by a reddish hue under fluorescent lighting. When CMV 2b is present, gene silencing is prevented, and cell death is inevitable. What we would like to see is if 2b can suppress silencing of GFP without the consequence of cell death. This will be done by concurrently introducing a cell death suppressor gene called AvrPtoB. The effect of AvrPtoB will be compared with a gene Ptoy207D, a mutant strain that triggers cell death upon over-expression of a particular gene (GFP). Other suppressors will also be examined against a control. We will compare the results to those obtained with resistant genes to determine if there is a difference between viral suppression and viral resistance mechanisms that are thought to be very similar or even the same by some investigators. However, we predict that the cell death pathway does not affect the gene-silencing pathway. We hope to see a fascinating outcome that supports the presented predictions.

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FREDRICK (FRED) LARABEE
Pacific Lutheran University, Tacoma, WA
In Dr. Yang's lab, we have been studying the role of Rop (RHO-like GTPase of plants) in cellular signaling. Rop is a small G-protein that acts as a molecular switch in many plant signal transduction pathways. We have been using the emerging technique of chemical genetics in order to discover previously unknown pathways that might involve Rop signaling. Chemical genetics uses small molecules to alter protein functions and is similar to traditional genetic approaches. The advantage to chemical genetics, however, is that genes whose mutation would normal kill the plant and hinder further study, can be altered without being lethal to the plant. In my project, we have been using a mutant of Arabidopsis that possesses a constitutive active (CA) form of Rop2, one of the eleven Rops in this plant. The constitutive active mutant has high levels of Rop2 in its active form, Rop-GTP. These CA-Rop2 plants have a characteristic cotyledon shape, different from the wild type, which can be used as a screen in the chemical genetic assays. By subjecting seedlings to a large library of chemicals and screening for chemicals that change the cotyledon shape back to the wild type, we hope to find chemicals that are involved in Rop pathways. Further study of these chemicals and their interaction with biological molecules will possibly help researchers uncover other Rop mediated signaling pathways.

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THOMAS LAUGHREY
San Bernardino Valley College, CA
My summer project in Dr. Nugent's lab involves the analysis of the TEN1 gene in the yeast, S. cerevisiae. Ten1p, Cdc13p, and Stn1p are an essential set of proteins the physically interact with one another and are critical for maintaining telomere integrity. The telomere is the end region of a eukaryotic chromosome. It aids in chromosome integrity and maintenance. A telemore's main function is to counteract the tendency of a chromosome to be shortened during replication. This summer I will attempt to isolate TEN1 mutant alleles and test them for temperature sensitivity. Having mutant TEN1 alleles that appear normal at permissive temperatures, but appear to be null-alleles at restrictive temperatures would present the opportunity for direct experimentation. Characterizing these mutant alleles will aid in the understanding of the TEN1 function. Also, it is known that Ten1p interacts with Stn1p and Cdc13p. This summer I intend to identify additional proteins that physically interact with Ten1p. This will be accomplished by use of the yeast-two hybrid assay and co-immunopreciptiation studies. I hope that my work will aid in understanding the molecular function of TEN1.

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HSIANG-I (JENNY) LEE
California State University, Long Beach, CA
My research in the Springer lab focuses on the shoot apical meristem (SAM). The SAM plays a vital role in plant development, which determines the shape and position of organs in plants. Unlike animals, plants form organs throughout their life. And it is this exciting difference that brings my attention to the study of genes that have impacts on plants. The Springer lab has been examining the LATERAL ORGAN BOUNDARIES DOMAIN (LBD) gene family specifically for its effects in the development of leaf formation. During the course of research, my goal is to functionally characterize LATERAL ORGAN BOUNDARIES DOMAIN36 (LBD36), which is closely related to LATERAL ORGAN BOUNDARIES (LOB) and ASYMMETRIC LEAVES2 (AS2), genes that are important for leaf development. The objectives of my project are to examine the expression pattern of LBD36 using promoter-GUS fusions, to analyze the phenotype of plants that gain and loss LBD36 function, and also to identify LBD genes in rice genome by using bioinformatics. The initiation of my project is to select the single copy T-DNA insertion of promoter LBD36:GUS fusion and to observe the phenotype of as2 and lbd36 EMS-mutants. Once the promoter LBD36:GUS fusion plants are collected, I will then apply the methods of GUS stain, histology analysis to analyze the expression pattern of LBD36. I will also look into ET1392, which is an enhancer trap line that has an insertion in LBD36 coding region. The dissection of the expression region will be done to verify expression pattern of ET1392. Besides the laboratory techniques that I will acquire, the use of bioinformatics will also be introduced, which allows me to inspect similarities of LOB domain genes between the Arabidopsis genome and the rice genome.

My overall research experience in the Springer Lab has further enhanced my ability in studying plant science. I appreciate the support from the members of the Springer Lab, and special thanks to Dr. Patty Springer for her patience and professional knowledge throughout the course.

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LINDA MORALES
University of California, Riverside
My research in the Bailey-Serres laboratory focuses on the development of new techniques for the analysis of gene expression. Gene regulation in plant cells occurs at the transcriptional level, as well as post-transcriptional levels, in individual cells and tissues. Most cells are interconnected by plasmodesmata that permit intracellular communication. Current methods for isolation of mRNA do not allow for purification on mRNA from individual cell types.. The goal of the NSF-sponsored project that I am participating in is to develop methods for purification of mRNA from specific cell types of plants. My goal is to construct a ribosomal protein P1 that has an additional His-FLAG tag (epitope tag) at either the amino or carboxy terminus. This gene will be driven by a CaMV (cauliflower mosaic virus) promoter in transgenic Arabidopsis thaliana. We will determine if this epitope tag permits affinity purification of ribosomes. My mentor, Eugenia Zanetti has already tagged several ribosomal proteins from the large and small subunit. I will assist here in the biochemical studies (purification of ribosomes) as well as examination of the effects of the tagged protein on plant phenotype. Tagged protein accumulation and incorporation into ribosomes will be assayed. If the tagged protein is assembled into ribosome, then ribosomes and/or polysomes with the tagged protein along with the associated mRNA will be extracted. Purification of the mRNA and use of microarray will be used as a tool to examine gene expression. If cell or tissue specific promoters are used gene expression in cell or tissue expression can be analyzed without disturbing intracellular communication.

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CATRINA ROMERO
California State University, San Bernardino
While in Dr. Borkovich's lab this summer I will be studying heterotrimeric G-proteins in Neurospora crassa. G-proteins are components of the signal transduction pathways for most developmental processes in this filamentous fungus. Our main interest in N. crassa heterotrimeric G-proteins is to study the Gβ subunit and Gγ subunit and to verify their interaction with one another. We will also study the interactions of Gβ and Gα subunits. The methods used for this study are the yeast two-hybrid assay and co-immunoprecipitation. For the yeast two-hybrid assay, we designed Gβ and Gγ primers based on cDNA sequences for these genes for N. crassa. We also designed vectors to transform yeast in order to study Gβ and Gγ binding. To study the Gβ and Gα relationship using the yeast two-hybrid assay, we used existing yeast strains and tested interactions using a filter assay for β-galctosidase. For co-immunoprecipitation we began by cloning FLAG, MYC, and HA tag sequences into an E. coli vector. The DNA was then purified from two transformants for each tag and the sent to the Genomics laboratory for DNA sequencing. The results showed the tags had been correctly cloned. The tag sequences are then going to be amplified using PCR, so that they can be cloned in large amounts into the caboxy terminus of the Gβ and Gγ genes. If time permits, we may be able to introduce another tag sequence, GFP, which will allow us to use fluorescence imaging to show the location of the Gβ and Gγ subunits in N. crassa.

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LUIS TORNER
University of California, Riverside
I am working in Dr. Linda Walling's Lab under the guidance of Virginia Alonzo. We are currently studying the role of proteins that stimulate prenylation reactions in Arabidopsis thaliana. These prenylation reactions lead to the biosyntheis of isoprenoids. Isoprenoids are compounds involved in plant growth and development. My project has three main goals that will partly be completed over the course of this summer and will be continued throughout the next academic year. I plan on aiding in the characterization of T-DNA insertion lines through the use of PCR. Once I characterize an insertion line that is homozygous, I will look for phenotypic variances. I will also attempt to determine the subcellular localization of prenylation stimulating proteins. In order to complete this part of the project I will need to make my own chimeric construct in which will contain a "reporter" gene such as GFP, YFP, or CFP. With the chimeric construct transformed into the plants, I will be able to see where the plant stores this protein at a subcellular level. Not only will I attempt to determine the protein localization subcellularly but I will attempt to determine tissue specific expression patterns of the proteins. This part of the project is very similar to the previously mentioned part, the only difference being that transgenic plants expressing promoter reporter genes will be evaluated. This summer I will construct promoter:GUS and promoter:GFP transgenes. With the identification of the subcellular localization as well as the tissue specific patterns and the information obtained by growing homozygous mutant lines, I will hopefully be able to contribute to the further understanding of these novel proteins.

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JACOB VASQUEZ
San Bernardino Valley College, CA
My summer research project in Dr. Raikhel's lab will address the question of how plants sense gravity. Plants can sense gravity, and consequentially roots grow down and shoots grow up. To further our understanding of this I will perform experiments on Arabidopsis, a model plant. We know that the endomembrane system is likely involved in the process of gravity sensing because mutations in proteins that are involved in vesicle transport and vacuole biogenesis (e.g. SGR3 and ZIG) alter gravitropic responses. We will use a genetic approach to find genes and proteins that are involved in the response to gravity. But instead of a "classical" genetic assay where changes in DNA are used to obtain mutants, we will employ a "chemical" approach. Chemical genetics uses small molecules to perturb, study and control cellular and physiological functions of proteins. When we find a "hit," that is, a chemical that does not allow a plant to respond to gravity, we can get the structure of the chemical that causes the agravitrophic phenotype. The long-term goal is to find the actual protein that is a target for the chemical compound; unfortunately, this question cannot be fully answered this summer.

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REBECCA (BEKI) MARTIN
New College of Florida, Sarasota, FL
My project in Cengiz Ozkhan's lab combines nanotechnology and plant biology. I am working with engineers to produce bio-conjugated fluorescent quantum dots to a lily flower stylar/stigma cysteine rich adhesion protein (SCA). Quantum dots are nano-sized semiconductor crystals made of cadmium and selenium. They fluoresce a variety of colors ranging from red to blue depending on their size. They are much more resistant to photobleaching than conventional fluorescent markers and their small size make them ideal for visualizing small proteins like SCA. SCA is important in the growth of lily pollen tubes within the style. Using quantum dots as fluorescent markers, it may be possible to localize SCA along a growing lily pollen tube and determine whether it is being taken up endocytosis or if it binds only to surface of a tube. Currently, we are working on stabilizing quantum dots in water and growing lily pollen tubes in vitro. Later, we will put them together with quantum dot labeled SCA in order visualize what happens. I am having a fantastic time learning about nano-technology and lily reproduction. I have been able to use and learn about, state of the art equipment, watch carbon nano-tubes being made and use Scanning Electron Microscopy to visualize things 10 µm and smaller. It is an amazing opportunity to integrate such different fields into an interesting and exciting project.

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