Center for Plant Cell Biology

REU 2004

REU Students and their Summer 2004 Research Programs

Undergraduate students were invited to apply to the Center for Plant Cell Biology (CEPCEB) to pursue individual research projects in the area of plant cell biology. In 2004, the following ten students were accepted from over seventy applicants who applied to this ongoing 10-week residential summer program.  Please click on the following student links to see photos and read about their Summer 2004 research programs in CEPCEB laboratories.

REU Student College/University CEPCEB Faculty Mentor
Marietta P. Boisdore Southern University at New Orleans Raikhel lab
Michelle Brown Mount San Jacinto Community College, CA Borkovich lab
B. Walter Evans University of Alabama Ding Lab
Candida S. Fielding Fort Valley State University, GA Bachant Lab
Ivann Martinez California State University, Long Beach Bailey-Serres Lab
Veronique Matthews Fort Valley State University, GA Yang Lab
Judy Ann Melendez Universidad Metropolitana, Puerto Rico Walling Lab
Jonathan Ringler Aquinas College, Grand Rapids, MI Eulgem Lab
Carrie Thurber Framingham State College, MA Judelson Lab
Justin D. Wood San Bernardino Valley College, CA Springer lab


Marietta P. Boisdore

The Raikhel laboratory is interested in understanding the role of vacuoles and the endomembrane system in plant growth and development. Mutations leading to a loss of function of genes encoding many of these components are either lethal because the genes are essential, or they have no effect on plant phenotype due to the presence of multiple genes with overlapping function. One new approach to identifying genes involved in endomembrane biogenesis is chemical genomics, in which a library of diverse chemicals is screened for compounds causing specific phenotypes. Using this approach the Raikhel laboratory has identified several novel drugs, namely Sortins 1 and 2, which affect vacuole biogenesis and root development of the weedy flowering plant Arabidopsis thaliana. My participation in this on-going research is to conduct experiments to identify the molecular targets of Sortin 1 using genetics. I will screen approximately 100,000 seedlings for those that are either resistant or hypersensitive to Sortin 1. As a convenient phenotype to detect mutants, I am working with a dose of Sortin 1 that inhibits root development. Thus, resistant mutants will have normal length roots in the presence of a high dose of Sortin 1, whereas hypersensitive mutants will have short roots in the presence of a non-inhibitory low dose of Sortin 1. Putative mutants will be confirmed in two ways: 1) Seeds will be germinated with and without Sortin 1 to confirm that the root phenotype is drug dependent; 2) Seedlings will be viewed by confocal microscopy to examine directly their vacuole morphologies. This is possible because the mutagenized plants also express a marker protein (╬┤TIP-GFP) that allows us to visualize the tonoplast in living cells. Mutants that are confirmed will be backcrossed by the Raikhel team in order to map and clone the genes responsible for resistance or hypersensitivity.

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Michelle Brown
B. Walter Evans

My project in Dr. Borkovich's laboratory involves the mutational analysis of two of the eleven putative hybrid histidine kinase genes of the ascomycete fungus, Neurospora crassa. Dr. Borkovich is conducting an on-going project to study the role of two-component regulatory systems in this multicellular fungus. These cascades regulate a diverse array of functions, ranging from responses to nutritional stress or chemical signals to multicellular development, chemotaxis and light sensing. Two-component systems consist of proteins containing histidine kinase and/or response regulator domains. The "knockout" gene constructs that I am using have already been made by Dr. Borkovich's team. My goal is to transform these constructs into the yeast, Saccharomyces cerevisiae, then into the bactierum Escherichia coli, and finally, into Neurospora crassa. As these transformations progress through a series of electroporations and analyses using PCR and Southern hybridization, my job is to identify and isolate the mutants for the putative histidine kinases, NCU 01823.1 and NCU 048341.1. By the end of the summer I hope to analyze the fungal mutants for cellular and developmental phenotypes, and their defects compared to those of the response regulator and histidine phosphotransferase protein.

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It has been my good fortune to be able to work in the Ding lab for the summer. The focus for this lab is on RNA interference (RNAi). RNAi is a conserved mechanism in which genes are silenced by mRNA degradation in a sequence specific manner. RNAi has been found to contribute to antiviral responses, development, and chromatin regulation in many different types of organisms. RNAi has also proven to be an important experimental tool for selected inhibition of expression of genes. My project is to determine if certain factors of a known mRNA degradation mechanism contribute to RNAi in fruit fly (Drosophila melanogaster) cells. For this project, I have been able to learn many knew ideas and techniques due to, in large part, the patience and kindness of my postdoctoral mentor, Dr. Saba Aliyari. I am very grateful for the chance to have these experiences and friendships received in this lab.




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In the Bachant lab, I am testing the idea that mutant strains of S. cerevisiae, that lack both the S phase and spindle assembly checkpoints, will be able to initiate and complete mitosis. RAD53 is a protein kinase that is the key regulator of the S phase checkpoint, which prevents mitosis when DNA synthesis is perturbed. rad53 mutants lack the arrest of mitosis, and the mutants move on through the cell cycle with unreplicated DNA. Our lab has observed that although rad53 mutants initiate mitosis, they do not complete it. Our hypothesis to explain this observation is that another checkpoint, the spindle assembly checkpoint, compensates for the loss of the S phase checkpoint. If so, we predict that cells containing mutations that cause both checkpoints to malfunction will start and complete mitosis even though DNA is not replicated. To test this I will generate and test two different mutant strains. (1) The first is a rad53 ipl1 mutant. IPL1 (Increase in PLoidy) is a protein kinase that is responsible for preventing chromosome segregation at the spindle assembly checkpoint in the event that the kinetochores are not properly attached to spindle poles. An ipl1 mutant is defective for this checkpoint and is thus unable to prevent chromosome segregation following spindle damage. (2) The second is a rad53 mad2 mutant. MAD2 (Mitotic-Arrest-Deficient) protein detects unoccupied kinetochores and arrests chromosome segregation if microtubules are not properly attached to the kinetochores. mad2 mutants are defective for this response, allowing chromosome segregation to continue even if chromosomes are not properly attached to the spindle. I will test these two double mutants to determine if they initiate and complete mitosis when DNA replication is perturbed.

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Candida S. Fielding
Ivann Martinez
Veronique Matthews
This summer I will participate in a project to determine the roles of four genes that each encode a protein of no known function that is highly up-regulated in response to low oxygen stress (hypoxia) in Arabidopsis thaliana seedlings. I will be learn how to grow Arabidopsis, perform stress treatments, extract and analyze DNA and mRNA, and evaluate data available from a number of web-based resources. Previous DNA microarray experiments in the JBS lab that compared wild type control and hypoxia treated Arabidopsis seedlings led to the identification of a group of gene transcripts that have increased abundance under hypoxia stress. Clustering of the DNA microarray data revealed a group of 216 genes that are induced under hypoxia stress; 109 of these genes encode proteins of unknown function. My project aims to identify the importance of four of these genes. My objectives involve: (1) The confirmation of data obtained by the DNA microarray experiment, by studying the changes in mRNA levels and their ribosome association after several time points of hypoxia treatment; (2) The identification of Salk T-DNA insertion alleles for each of these genes, to study whether a loss of each of these genes affects survival of the stress. This will involve the identification of individual plants that are homozygous for the T-DNA insertion allele, from a family of plants that are segregating for the insertion mutation. Screening and genotyping will be done by use of PCR; (3) The compilation of publicly available data on these four genes. Hypoxia stress treatments on wild type and mutant lines will be used in order to compare their response phenotypes.

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Our lab focuses on the role of Rop GTPase signaling networks in the establishment of cell polarity. I will investigate mechanisms for cell morphogenesis in the epidermal cells of the Arabidopsis thaliana leaf. These epidermis cells have a puzzle-like shape. The formation of the epidermis cells requires coordination and communication between adjoining cells. For this reason, Arabidopsis leaf cells serve as a model system to investigate the mechanism of interdigitated cell formation in a multicellular organ. Dr. Yang's lab identified 11 Arabidopsis genes that belong to the RIC family (Rop-interacting CRIB-motif containing proteins) that interact with ROP GTPase in the process that interconnects epidermal cells. ROP activates RIC4, which promotes cortical fine F-actin required for outgrowth of lobes. At the same time ROP inactivates RIC1, which promotes transverse microtubules that inhibit outgrowth in the indentation region of the cell. The goal is to figure out how RIC1 proteins promote microtubule assembly. To investigate which domains of the protein are important for microtubule binding and promoting activity, I will perform the polymerase chain reaction (PCR) and plasmid cloning to generate deletion mutants. Next, I will use the gene-gun method to transfer the plasmid DNA into the leaf cells. Then, I will use confocal microscopy to observe how the protein localizes within the cell and function in microtubule assembly. These along with other techniques of molecular biology, biochemistry, and cell biology will be involved in this investigation.

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Judy Ann Melendez

In Dr. Linda Walling's lab I am screening a combinatorial chemical library to identify molecules that specifically inhibit or activate leucine aminopeptidases (LAPs), which catalyze the hydrolysis of amino acid residues from the amino terminus of proteins. LAPs are hexameric metallopeptidases that have alkaline pH optima and are inhibited by the potent aminopeptidase inhibitors amastatin and bestatin. The tomato LAP-A is the best biochemically characterized aminopeptidase in plants. LAP-A is highly expressed at the RNA and protein level in response to wounding, various biotic and abiotic stresses and during both floral and fruit development. The plant model organism, Arabidopsis thaliana, has three LAP enzymes, but the roles of these enzymes are unknown. Last summer an NSF-REU student developed a chemical genetics procedure to identify small molecules that inhibit or enhance the activity of tomato LAP-A and Arabidopsis LAP-1. This summer I hope to further characterize tomato LAP-A and Arabidopsis LAP inhibitors and continue to screen the chemical library for new inhibitors and activators. The goal is to find small molecules that specifically inhibit and activate LAPs and not other classes of aminopeptidases. This will provide the Walling lab with tools to better understand the function and importance of LAPs in plants.




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Jonathan Ringler
Carrie Thurber

My lab project involves one of the largest factors in natural selection, the ability of a species to overcome pathogens. Plants are no exception to this rule, though it may seem as if they are at a disadvantage. The immunities of the plant cell ultimately work off of two basic principles. The first recognizes that a pathogen that cannot enter cannot infect; ergo the utilization of complex cell walls and membranes. The second maintains that a pathogen cannot grow in a toxic habitat. When a plant cell senses a pathogenic breech, it initiates a doomsday sequence; the cell becomes poisoned and cannot support the invading threat.

The genetic mechanisms controlling the suicide are currently the subject of research in the Eulgem lab. Two genetic families have been targeted for investigation. One consists of genes that code for calcium-binding proteins (CaBPs) and appear to be thrown into full gear during invasions. The other, known as the WRKY group, seems to be integral in many functions, including immunities. Examining members of both families may expose an individual cell death sequence. The investigation of these functions involves inserting a piece of foreign "T-DNA" by a plant infecting bacterium into a targeted gene sequences, thereby nullifying previous wild-type functions. Another method involves reporter genes, specialized sequences that are inserted at the end of particular gene codes and made manifest when the gene is transcribed. My mentor and I are using these tools to observe the functions of the aforementioned gene families, which may be involved in the plant immune system.

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Dr. Judelson's lab studies the genetics of the spore-producing oomycete Phytophthora infestans. P. infestans is a fungus-like organism that produces spores (sporangia) that are uncharacteristic of most true fungi. For example, the Phytophthora sporangia are undesiccated and release zoospores. These zoospores are the main mechanism by which P. infestans spreads disease among plants. Potatoes infected with P. infestans, a condition termed potato late blight, die quickly and spread the disease across large regions in a short period of time. This disease was the major cause of the 1845 potato famine in Ireland and has since resurfaced in potato crops worldwide. I will initially use bioinformatic tools to study genes previously found to be up-regulated during sporulation in this oomycete. These genes include P. infestans cleavage genes, termed Pic genes, and P. infestans sporangia genes, termed Pisp genes. BLAST, or Basic Local Alignment Search Tool, will be used to compare the P. infestans genes to those of related Phytophthora species; P. sojae, the cause of soybean root rot, and P. ramorum, the cause of sudden oak death. By comparison of the conserved regions of these genes it is my goal to determine the functionally important regions of promoters of selected genes. These regions will be amplified using PCR and clone into a plasmid vector. If time permits, this vector will be attached to a GUS reporter gene and tested in P. infestans.

The LATERAL ORGAN BOUNDARIES (LOB) gene is expressed in the boundary found between lateral organs and shoot apical meristems of plants. LOB is a member of a large gene family called the LATERAL ORGAN BOUNDARIES DOMAIN (LBD), which consists of forty-three similar genes that are found only in plant species. My research will focus on the functional analysis of members of the LBD gene family in Arabidopsis thaliana. To understand the function of these genes, three approaches will be used. (1) The expression pattern of LBD25, one member of the LBD gene family, will be studied. I will analyze transgenic plants that contain a GUS reporter gene under the control of the LBD25 promoter to determine the developmental expression pattern of LBD25 in plants and plant tissue sections. (2) Loss-of-function mutants will be analyzed. We already know that the lbd25 single mutant displays no visible phenotype. This suggests that the function of LBD25 may be redundant to other LBD genes. To test this, plants are mutant for both lbd25 and the related gene ASYMMETRIC LEAVES2 (as2) will be examined. I will use PCR to identify the double mutant plants (lbd25, as2) from a segregating population. The double homozygotes will be examined for phenotypes not present in either single mutant. (3) Publicly-available microarray data will be analyzed to obtain information about LBD gene expression and the processes in which LBD genes may control.



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Justin D. Wood

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University of California, Riverside
900 University Ave.
Riverside, CA 92521
Tel: (951) 827-1012

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Center for Plant Cell Biology
Botany & Plant Sciences Department
2150 Batchelor Hall

Tel: (951) 827-7177
Fax: (951) 827-5155
E-mail: genomics@ucr.edu