UCR

Center for Plant Cell Biology



REU 2005


REU Students and their Summer 2005 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 2005, 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 2005 research programs in CEPCEB laboratories.

REU Student College/University CEPCEB Faculty Mentor
Benjamin Becerra California State Polytechnic University, Pomona, CA Smith Lab
Michelle Brown Mount San Jacinto Community College, CA Springer Lab
Hilary Christensen Carleton College, MN Pirrung Lab
Robert Dick Iowa State University, IA Zhu Lab
Candida Fielding Fort Valley State University, GA Bachant Lab
Janet Lee University of California, Los Angeles, CA Jin Lab
Noelle Oas Saint Mary's University, MN Eulgem Lab
Amy Sainski University of Wisconsin, Parkside Raikhel Lab
Ricardo Sayegh Chaffey Community College, CA Girke Lab
Quynh Vu University of Houston, TX Borkovich Lab
Naa-Darkua Wellington Xavier University, LA Gallie Lab

 

 

BENJAMIN BECERRA
CAL STATE POLYTECHNIC UNIV., POMONA, CA
BecerraB

Dr. Smith's laboratory is focused on understanding the molecular mechanisms that control the specification of flowers during inflorescence development in the shoot apical meristem (SAM) of Arabidopsis plants. As simple as it may sound, flowering is actually one of the most dramatic and important developmental events that occurs during the lifetime of a flowering plant. However, very little is known about the cellular and molecular events associated with flowering. Previous research by Dr. Smith and his colleagues has demonstrated that the two BEL1-like (BELL) homeodomain transcription factors PENNYWISE (PNY) and POUNDFOOLISH (PNF) interact with another transcription factor SHOOTMERISTEMLESS (STM) to form a heterodimer. Genetic studies suggest that inflorescence development requires the redundant activities of PNY-STM and PNF-STM heterodimers, which function to specify flowers, regulate early internode patterning events and maintain the boundary between initiating floral primordia and the inflorescence meristem. Currently, Dr. Smith's laboratory is focused on understanding the molecular mechanisms by which these homeobox transcription factors specify floral cell fate in response to environmental and endogenous cues.

My goal as an REU student is to characterize the expression patterns of genes regulated by PNY-STM and PNF-STM heterodimers, identify and characterize mutations in pny pnf plants to suppress the non-flowering phenotype and map the genomic positions of these mutants. The laboratory methods I will be utilizing throughout this summer include molecular techniques such as activation tagging, DNA extraction, PCR, gel electrophoresis, and DNA sequencing. I will use histological approaches to localize promoter GUS expression patterns in the SAM and quantitate developmental phenotypes that disrupt flowering and inflorescence architecture in Arabidopsis.

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The summer project in the Springer lab is to investigate whether there is a connection between the LATERAL ORGAN BOUNDARIES (LOB) gene and brassinosteroid signaling in Arabidopsis thaliana. The LOB gene is expressed in the boundary between the meristem and the lateral organs. While the exact function of the LOB gene is unknown, LOB is known to interact with a subset of transcription factors. Some of these transcription factors are regulated by brassinosteroids (BR). Brassinosteroid signaling is associated with many developmental and physiological processes, such as germination, hypocotyl elongation, and wound response.

Interestingly, when LOB is over expressed, the dwarf phenotype that results is very similar to the phenotype of an Arabidopsis thaliana plant that is brassinosteroid deficient. Since root and hypocotyl elongation are indicators of the BR response, precise measurements of both will be taken when plants that over express LOB are exposed to varying concentrations of brassinolide (BL), the most biologically active of the brassinosteroids. In addition, assays to examine molecular responses to BR will be done following BL application in over expressed LOB plants and in wild type plants. These experiments will help to clarify the connection between the LOB gene and brassinosteroid signaling.

 

 

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MICHELLE BROWN
MOUNT SAN JACINTO COMMUNITY COLLEGE, CA 
BrownM
HILARY CHRISTENSEN
CARLETON COLLEGE, MN

Small RNAs in Arabidopsis play important roles in gene expression regulation. They consist of two main classification groups: Micro RNAs, which originate from RNA hairpins, and Short Interfering RNAs, which are cut from longer sections of double-stranded RNA. Current methods for detecting these small RNAs are not very effective, so the goal of this study is to develop a method of small RNA detection using a microarray analysis. Consisting of complementary surface-bound primers that fluoresce when they hybridize to their target RNAs (or cDNAs), microarrays are already widely used. However, the small size in the case of microRNAs or siRNAs (20-23 nucleotides) can result in problems such as weaker hybridization between primer and target. The Pirrung lab has developed a technique called APEX (Arrayed Primer EXtension) to specifically analyze RNAs. The microarrays are prepared with complementary primers that hybridize to the RNAs of interest, after which reverse transcriptase adds a fluorescent dideoxy nucleotide. Previous studies have shown that chemically modified primers have a higher affinity for RNA, specifically a 2' O-Methyl group or a 2' Fluorine. 2'F is more suitable for high-density microarrays since the extra methyl group on the primer renders it unrecognizable by the enzymes. Due to the fact that only the pyrimidines (C and U) are available commercially in fluorinated forms, our targed RNAs will be A and G rich in order to allow the presence of as many fluorinated bases in the complementary primer as possible.

 

 

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I'm working in Jian-Kang Zhu's lab to understand how plants respond to environmental stress. By identifying mutant phenotypes in Arabidopsis and what genes are responsible for the observed mutant phenotypes we can generate hypotheses about what roles these genes play in the plant stress response. The Zhu lab has isolated mutants with altered expression of the NCED3 gene, which encodes a key enzyme in abscisic acid (ABA) synthesis. I am working with the NCED3 deregulated1 (ced1) mutant, which has increased ABA accumulation and reduced growth under stress conditions. By constructing a double mutant of ced1 with the ABA-deficient mutant (aba2-1), I will assess whether the increased ABA content of ced1 is required for the stress-sensitive phenotype. I will also undertake additional physiological characterization of the ced1 mutant.

I am also working to identify the mutated gene in the repression of silencing2 (ros2) mutant. In ros2, a normally active RD:29A:LUC transgene construct as well as the endogenous RD29A gene are silenced. Mapping of ros2 has narrowed the location of the mutation to a defined interval. I will sequence ros2 candidate genes in this interval in order to locate the mutation. My work this summer will be under the direction of Dr. Verslues in Dr. Zhu's lab.

 

 

 

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ROBERT DICK
IOWA STATE UNIVERSITY, IA
CANDIDA FIELDING
FORT VALLEY STATE UNIVERSITY, GA

In the Bachant lab, I am trying to understand a possible role for SUMO modification in histone acetylation, DNA replication, and genome stability. SUMO is a small protein belonging to the ubiquitin family of protein modifiers. The addition of SUMO can modulate the ability of proteins to interact with their partners, alter their patterns of subcellular localization and control their stability. SUMO is synthesized as a precursor and processed by hydrolases to make the carboxy-terminal diglycine motif available for conjugation. It is then conjugated to proteins by means of E1 activating, E2 conjugating, and E3 ligating enzymes. Yet after conjugation, SUMO can be removed by a class of enzymes known as SUMO isopeptidases. The main isopeptidase in budding yeast is Smt4. Smt4 is required for budding yeast cells to be able to withstand conditions where hydroxyurea, an inhibitor of DNA replication, is present. This observation suggests that when SUMO modification is misregulated, DNA replication errors arise at a high frequency and must be appropriately processed by the repair machinery in order for the cell to survive. A dosage suppressor screen performed on a Δsmt4 strain has shown that elevated levels of HOS3, which is a gene encoding histone deacetylase, has the ability to suppress smt4 mutant's sensitivity to hydroxyurea. Histone acetylation itself is very important to the experiment being conducted because this process correlates with transcriptional activity in many genes, including those involved in origin of replication firing. The knowledge of the relationship between SUMO modification, and histone acetylation/chromatin structure brings us to the project of understanding how HOS3 suppresses the hydroxyurea sensitivity of smt4 mutants. The questions that we are addressing to help us further our understanding are as follows: Does HOS3 suppress other smt4 mutant phenotypes, are acetylated histone levels affected by the loss of Smt4 function, and is origin firing altered in smt4 mutants.

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Dr. Jin's lab is interested in the mechanisms of plant pathogen resistance. Plant diseases cause significant crop losses each year. The pathogen P. infestans causes late blight, a disease affecting potato and other Solanaceous plants. The RB gene provides a broad-spectrum resistance to late blight and has recently been isolated. EDS1, NDR1, SIPK, and WIPK are genes that are important signaling components in the R gene-mediated disease resistance pathways. In order to determine if these four genes also play important roles in the RB resistance pathway, my project this summer is to study these genes using virus-induced gene silencing. The silencing construct containing the gene of interest is first transformed into A. tumefaciens. I will then inoculate N. benthamiana with the agroinoculum. The virus carrying the silencing gene fragment is extracted from the infiltrated leaves after eight days post silence induction. The extract sap is then used to inoculate wild potato plants that carry the RB resistance genes. After the gene of interest has been silenced, I will use a detached leaf assay to test for resistance. My project will contribute to a better understanding of the RB-mediated resistance signaling pathway, which will hopefully one day lead to crops with improved pathogen resistance traits.

 

 

 

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JANET LEE
UNIVERSITY OF CALIFORNIA, LOS ANGELES 
NOELLE OAS
SAINT MARY'S UNIVERSITY, MN

One of the largest threats to the agricultural industry is disease susceptibility of many crop species. Over the past decades, a comprehensive research effort has been conducted to understand the means by which plants defend themselves against pathogens. Major advancements have been made in this area, such as the identification of resistance genes (R-genes) and components of defense signaling pathways. However, mechanisms of transcriptional regulation associated with these pathways are not well understood. The Eulgem lab is interested in the molecular mechanisms which control transcriptional reprogramming triggered during the immune response of plants. For this purpose the model system Arabidopsis thaliana Peronospora parasitica is used.

This summer, I will be working with several short-conserved promoter motifs of co-regulated defense genes previously identified in the lab. These conserved motifs may constitute cis-elements important to coordinate expression of defense genes. The ability of these motifs to bind with nuclear proteins present in Arabidopsis thaliana will be tested using Electrophoretic Mobility Shift Assays (EMSA). For the EMSA, the motifs will be labeled with radioactive phosphate (32P) and incubated with nuclear protein extracts before being subjected to gel electrophoresis. If the motif is able to bind to the nuclear proteins, the migration of the complex will be slowed during electrophoresis, and there will be a shift in the position of the band. If a shift in band location is observed, a yeast one-hybrid system can be used to identify cDNAs encoding transcription factors that interact with the respective conserved promoter motif.

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This summer I am privileged to be working in Dr. Raikhel's lab using chemical genomics to study vacuolar biogenesis of Arabidopsis cells. There are three main experiments I will be contributing to this summer, all involving chemicals previously screened and found to have varying effects on the Arabidopsis endomembrane system. The first experiment involves isolating mutant plants based on their sensitivity to the chemicals. I will be screening an EMS-mutagenized population to identify plants that are hypersensitive to the chemical 247 (ID number from Chem Library 5850247). Once hypersensitive mutants have been identified, the lab can use map-based cloning to discover where the mutation exists and what pathways the chemical affects. The second experiment also tries to find what pathways are influenced by the chemicals. Here we will be treating wild type seedlings with chemicals for short periods of time. We will then extract RNA and analyze the accumulation levels of different transcripts using RT-PCR. The genes to be monitored were chosen from previous micro array experiments. Finally, the goal of the third experiment involves the characterization of the effects of the chemical on several florescent markers using a confocal microscope. The markers will label plant proteins that, under normal conditions, are targeted to specific organelles, such as the nucleus, ER, golgi body, and tonoplast.

 

 

 

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AMY SAINSKI
UNIVERSITY OF WISCONSIN, PARKSIDE
RICARDO SAYEGH
CHAFFEY COMMUNITY COLLEGE, CA

The Girke lab is interested in improving the current models of genes with unknown function that are encoded in entirely sequenced plant genomes. Those annotation improvements can often provide important leads for the functional identification of unknown genes on the molecular and biological levels. In this project I will use a comparative genome analysis approach to identify incomplete and new genes in the genome sequences from Arabidopsis and rice. The following three approaches will be used to achieve this goal:

1) Assuming there is a low degree of gene order similarity between rice and Arabidopsis (synteny), the identification of identical gene pairs can indicate that the current genome annotation has falsely annotated one longer gene as two separate ones. In those instances the two truncated genes will be merged into one and the correctness of the resulting protein sequence will be confirmed by comparisons against the UniProt databases.
2) Completely unidentified genes will be detected by searching for similarities between the intergenic regions and the UniProt databases. Significant similarities in this analysis will result in the discovery of new genes in the annotations of the two genomes.
3) Incomplete genes will be identified by comparing the similarities of the intergenic and protein coding regions from the two organisms with the UniProt database. If intergenic and coding segments of the same gene show similarity to one protein then this indicates a truncated gene model that may be correctable with the generated similarity information.

The following computational tools will be used to automate the required searches and analysis steps for this project. Large scale sequence similarity searches of ten thousands of sequences will be performed with the BLAST program on the command-line. Perl scripts will be written to parse and analyze the results. Statistical analysis will be performed with the R program and the final results will be uploaded into an existing web-based database.

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Dr. Borkovich's lab is part of an NIH-funded project along with labs from UCLA and Dartmouth to understand more about functional genomics in Neurospora crassa. Approximately 10,600 N. crassa genes are present in the genome, but their functions have yet been studied. My objective for this summer project is to find putative transcription factor(s) that can be regulated by G proteins and other upstream factors. This will be done by analysis of phenotypes exhibited by G protein mutants, and genetic and molecular analysis to verify their epistatic relationships. Under my mentor, Gyungsoon, I will test the phenotypes of 99 transcription factors and G-protein mutants for growth, female fertility, and conidiation. For this, several tests will be conducted. First, fresh transcription factor mutants will be grown in various types of media for analysis. Then, phenotypes of each will be studied and compared. Thirdly, candidates from 99 transcription mutants will be screened and further examined against G-protein mutants. Lastly, epistatic relationships will be determined using data gathered from above experiments as well as molecular data taken from Northern analysis or RT-PCR. By the end of this project my goal is to find the downstream transcription factor(s) of G-protein signaling in Neurospora crassa.





 

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QUYNH VU
UNIVERSITY OF HOUSTON, TX
NAA-DARKUA WELLINGTON
XAVIER UNIVERSITY, LA

Excessive light in plants leads to the augmented production of reactive oxygen species. This activity results in oxidative stress and the damage of many of the cellular components in plants. Plants are able to deal with the stress through an oxygen radical scavenging system in which ascorbic acid plays a key role. Asc is also known as vitamin c and it is the most abundant antioxidant found in plant chloroplasts. Asc works by reducing hydrogen peroxide either directly or through ascorbate peroxidase (APX). The product monodehydroascorbate (MDHA), a very unstable molecule then dissociates instantly to either Asc and dehydroascorbate (DHA) or is reduced to Asc by the enzyme MDHAR. DHA is either reduced to Asc by the enzyme dehydroascorbate reductase (DHAR) or undergoes irreversible hydrolysis to 2,3-diketogulonic acid. Plants with increased expression of DHAR have more Asc and are resistant to oxidative stress.

For this project I will be determining the functions Asc plays in plant photosynthesis and photoprotection using transgenic tobacco plants that have altered DHAR expression. The western-blot method will be used to screen DHAR overexpression and DHAR RNAi plants. I will analyze the photosynthetic performance such as carbon dioxide assimilation rate and transpiration rate of control, DHAR overexpression and DHAR RNAi plants using Li-Cor Gas Analyzer. Finally I will study the light and carbon dioxide response combined with chlorophyll fluorescence measurements to investigate the efficiency of light and CO2 of these transgenic plants.

 


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E-mail: genomics@ucr.edu

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