Research Experiences for Undergraduates (REU Program)
June 16- August 22, 2003
Overview:
An NSF-Sponsored Program to Provide Undergraduates Research Experiences
in Plant Cell Biology
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. This year, eleven students were
accepted to this ongoing 10-week residential summer program - an
increase over last summer where eight students participated.
Each student has a faculty and a graduate or postgraduate mentor.
In the initial week of the program, students were introduced to
the basics of plant cell biology as well as developing areas in
plant cell biology in which UCR has expertise, including genomics,
proteomics and bioinformatics, through a series of lecture/laboratory
exercises. Students will then spend nine weeks on a research project
of their choice. To further enrich the students and to guide them
toward graduate studies, students will participate in workshops
to enhance learning skills and professional development, and to
discuss ethics in science.
The schedule of events for the 10-week session is as follows:
Week One: Attend a week of lecture/labs to become
oriented to the program and to pick a research project for in-depth
study.
Week Two- Nine: Pursue individual research projects.
Attend weekly lab meetings with the other awardees. Attend
weekly CEPCEB research presentations.
Week Ten: Complete a write-up of the laboratory project.
Present a 15-minute talk detailing the results of the project.
Undergraduate students who participate in the REU program have
to be enrolled in a two- or four-year college, and be citizens or
permanent residents of the U.S.A. Students were expected to
have completed one year of Chemistry and Biology in preparation
for this program.
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Faculty
The awardees have the opportunity to work with the following members
of the Center for Plant Cell Biology. The area of research
in each laboratory is indicated. Please follow the links to
the members' web pages to further explore their research areas.
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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 |
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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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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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Kim
Carpenter
Western Washington Univ., Bellingham, WA |
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Marissa
Faeidan
University of California, Riverside |
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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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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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Fredrick
(Fred) Larabee
Pacific Lutheran University, Tacoma, WA |
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Thomas
Laughrey
San Bernardino Valley College, CA |
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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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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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Hsiang-I
(Jenny) Lee
California State University, Long Beach, CA |
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Linda
Morales
University of California, Riverside |
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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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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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Catrina
Romero
California State University, San Bernardino |
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Luis
Torner
University of California, Riverside |
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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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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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Jacob
Vasquez
San Bernardino Valley College, CA |
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Rebecca
(Beki) Martin
New College of Florida, Sarasota, FL |
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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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