UCR

Center for Plant Cell Biology



REU Students and their Summer 2017 Research Programs


Summer 2017 REU Students and their Research Projects

 

The need for scientists in plant biology-related disciplines is growing. The converging global challenges of increasing demand for food, the need for renewable energy, environmental degradation, and limited natural resources require science-based solutions developed by appropriately trained plant scientists. Most life-science disciplines are experiencing a major shift towards the use of innovative high-throughput technologies. We think it is of critical importance that life-science undergraduates get exposed to this new style of biological research. The NSF-funded research experiences for undergraduates (REU) program hosted by the Center for Plant Cell Biology (CEPCEB) at UC-Riverside is focused on next-generation biology of plants and plant pathogens. Each summer 10 – 14 students from US colleges and universities are invited to pursue individual research projects in the areas of cellular and molecular biology of plants and their pathogens. The CEPCEB-REU targets a broad range of students from both two- and four-year colleges, including institutions that offer limited research opportunities or serve groups traditionally underrepresented in the sciences. Program participants get exposed to innovative experimental techniques and benefit from state-of-the-art genomics, bioinformatics, microscopy and proteomics facilities. In summer 2017, 13 students were selected for this ongoing 10-week residential summer program. Please click on the following student links to see photos and read abstracts about their summer 2017 research projects in UCR laboratories.

 

 2017 CEPCEB REU students

 group

 

 2017 CEPCEB REU students

REU Student

College/University

Lab/PI

Sabrina Bimson

Ursinus College

Julia Bailey-Serres

Cora Bright

University of North Carolina at Pembroke

Venu Reddy

Selena Burke

Frostburg State University

Daniel Koenig

Emmanuel Cuevas

California State University Long Beach

Paul Nabity

Nicholas Curtis

University of Texas at Austin

Ian Wheeldon

Hannah Hall

Middle Tennessee State University

Jaimie Van Norman

Zachariah Jaramillo

Brigham Young University

Thomas Eulgem

Joshua Johnson

Central College

Kathy Borkovich

Vanessa Peterson

California Polytechnic State University, San Luis Obispo;

Zhenbiao Yang

Tamera Taylor

Chaffey Community College

Xuemei Chen

Ngoc  (Iris) Thai Hieu Tran

Riverside City College

Meng Chen

Aeriel Vanluesauls

Riverside City College

Katy Dehesh

James Jen Yen

UC Riverside

Eugene Nothnagel

 Sabrina Bimson, Ursinus College, (Bailey-Serres lab)

sabrina

 Translatome Analysis of the Phosphate Starvation Response of Oryza sativa

The human population is predicted to reach ten billion over the next three decades. This estimate coupled with climate change will demand greater and more effective agricultural output as changes in arable land occur. In order to sustain crop yield, food production requires application of fertilizers containing phosphorus on agricultural fields. Modern agriculture largely depends on phosphorus derived from phosphite rocks. Phosphorus current global reserves may be depleted in 50-100 years. This rapid decline is predicted to produce a significant rise in prices that may make this fertilizer unaffordable to poor farmer’s communities. Consequently, farmers who cannot afford phosphorous fertilizer will struggle to grow rice in low phosphate conditions. This project aims to find genes critical to the low phosphate survival, a signaling network that reorganizes root and shoot development to maximized phosphate absorption and utilization.  Specifically, rice (Oryza sativa cv. Nipponbare) will be grown with or without a phosphate source, and the root/shoot tissue will be harvested for mRNA and phosphate level analysis. Techniques used for mRNA analysis include Real-Time Polymerase Chain Reaction (qPCR), Translating Ribosome Affinity Purification (TRAP), RNA-Sequencing, and confocal microscopy. By use of these tools we will decipher the regulation of genes at different levels of gene expression, between transcription and translation, as well as within specific cell-types of roots and shoots. The effective long-term goal is to grow sustainable rice in low-phosphate conditions to the same level of rice grown with an exogenous phosphate source enhancing low input production of the major source of carbohydrates for half the world. 

 

Cora Bright, University of North Carolina at Pembroke (Reddy lab)

cora

 Genome Wide Quantification and Analysis of Master Plant Regulator WUSCHEL/DNA Interaction

Plant growth above ground is regulated by a structure called the Shoot Apical Meristem (SAM) which contains a group of undifferentiated cells that must be constantly maintained to ensure a balance is kept between their undifferentiated identity and fully differentiated organs. The WUSCHEL (WUS) protein is essential in meristem activity in Arabidopsis thaliana as it works by regulating several key components of the SAM molecular network.  In previous studies, when WUS is mutated to an inactive form, no meristem is formed at all. WUS upregulates and is in turn negatively regulated by CLAVATA3 (CLV3) forming a feedback loop that is a critical component of meristem growth. While WUS activity relating to individual targets has been investigated, a more comprehensive inquiry into its full range of targets would be helpful to characterize the nature and role of this factor. The first focus of this study is on large-scale patterns of DNA binding interaction with WUS in Arabidopsis. This will be done through gathering and analysis of ChIP-Seq data from Arabidopsis inducible LER p35S:WUS-GR ap1-1 cal-1 plants and pure ap1 cal-1 control plants, allowing for an accurate comprehensive in vivo snapshot of WUS activity throughout the genome. The second scope of this study will be looking at the transcriptional regulation events surrounding a WUS forced dimer that will be introduced in plants in a variety of different conditions. This will open different avenues for tracking the forced dimer itself and the different phenotypes produced by the WUS dimeric protein over time.

 

Selena Burke, Frostburg State University (Koenig lab)

 selena

 Local Adaptation of Barley to Climatic and Competitive Environments

Rapid, global climate change is one of the most urgent threats to our natural and agricultural systems. It is imperative that we understand how organisms can survive in vastly different environments to combat the negative consequences of climate change. Beginning in 1927, Harlan and Martini initiated a series of evolutionary experiments to explore the genetic basis of local adaptation in an agricultural context. They intercrossed barley lines collected from diverse environments and competed the progeny of these crosses against each for nearly half a century in two vastly different climatic environments (Bozeman, MT and Davis, CA). We will compare material from each of these conditions to search for the genetic and phenotypic basis of adaptation to climate. We will first compare the performance of barley landraces, as measured by seedling growth rate, under conditions mimicking the climates found at both sites in 14 day trials. Subsequent experiments will compete early and late generations of Bozeman and Davis adapted populations under greenhouse conditions in a six week trial. In parallel, we will use molecular techniques to explore the genetic changes in each population over time to search for alleles that may have contributed to local adaptation. These experiments will provide insight into the genetic basis of adaptation to local environments, and will elucidate how standing genetic variation in a composite cross population impacts overall plant growth.

 

Emmanuel Cuevas, California State University Long Beach (Nabity lab)

 emmanuel

 Woolly Apple Aphid resistance in a variety in apple plants

The Woolly Apple Aphid (WAA) is an insect that is native to Eastern North America but has been able to spread world-wide. Apple trees are its main source of food and it mainly feeds on wounds, cuts and axils of the tree. When the Woolly Apple Aphid feeds on apple trees it reduces the quality and amount of the apples by reducing the nutrient movement in the tree. In this study, we tried to find out the fecundity and death rate of the Woolly Apple Aphid in various types of apple plants. The plants vary in resistance to the WAA and the experiments will be held in a greenhouse in Riverside California. After assessing the WAA, the data will be analyzed using R a statistical programming language. Another form to understand the plant defenses when they are exposed to the WAA is Callose staining which will be performed to see the differences in the resistance response of various types of apple plants. The plants 02-O3R5-001, 02-O3R5-099, Geneva 935 and Geneva-16 will most likely be the most susceptible to aphid attack and will show the greater aphid colony growth. It is important to understand how the WAA infests various apple trees because it is a great source of food around the world. By understand why the WAA attacks the plants and how we can stop it we can increase our food production going into the future.

  

Nicholas Curtis, University of Texas at Austin (Wheeldon lab)

 nicholas

 Engineering gene expression in Yarrowia lipolytica using synthetic activators

Yarrowia lipolytica is an industrially relevant oleaginous yeast known for its ability to produce and accumulate large quantities of lipids. Understanding mechanisms of gene activation allows a greater utilization of Y. lipolytica as a host for production of diverse oleochemicals. To engineer and tightly control gene expression for pathway engineering, we will study the impact that different activation domains have on gene transcription. The selected activation domains will be fused to a DNA-binding domain to create a synthetic transcription factor, and its impact on the expression of hrGFP will be tested. The effect of changing the number of binding motifs in the synthetic promoter will also be investigated. Once an effective activation domain has been identified, a CRISPR-dCas9 activator system will be employed to study the impact of different activation regions on the expression of downstream proteins. By choosing different sgRNA targets, we will use a dCas9-activation domain fusion to activate fluorescence in a synthetic and using several native promoters. Using this developed CRISPRa system, production of industrially-relevant products will be engineered and optimized in Y. lipolytica.

 

Hannah Hall, Middle Tennessee State University (Van Norman lab)

 hannah

 Identification of PLK1 protein domains important for polar localization

Polarly Localized Kinase 1 (PLK1) is a transmembrane receptor-like kinase (RLK) with a known developmental tissue patterning in roots, unlike other RLKs that play roles in developmental patterning that remain uncharacterized. PLK1 fused with green fluorescent protein (GFP) shows lateral polarity in root tissue, but protein directionality differs based on cell type. The conditions under which this directionality is maintained disrupts other transmembrane proteins’ localization, even disrupting PLK1 localization to plasma membrane regions in mutant Arabidopsis roots. This suggests that PLK1 localization to specific regions depends on cell-to-cell communication, but not negating the importance of specific domains of PLK1 protein for localization to the correct regions. For the experiment sets of truncated PLK1 proteins with missing domains at the C-terminal end were used. Using the laser scanning confocal microscopy (Leica SP8) the localization of these truncated version of PLK1 fuse to GFP were examined and compared to the full-length PLK1:GFP fusion in mutant and non-mutant roots. Plants were also treated with pharmacological inhibitors of protein trafficking or cytoskeletal elements to determine if localization could be disrupted. These data will provide insight on how PLK1 is directed to specific plasma membrane regions and allow for comparisons with existing membrane protein localization paradigms.

 

Zachariah Jaramillo, Brigham Young University, (Eulgem lab)

 zachariah

 From Error to Advantage –

How Arabidopsis thaliana has harnessed transposon insertions in order to regulate localized cell death during immune responses

Plants, like all living organisms, are constantly exposed to disease-causing pathogenic microbes. In response to this selective pressure, plants have developed an intricate immune system that allows them to detect unique chemical signatures and then respond in a race-specific manner to an invading pathogen.

 Hyaloperonospora parasitica arapidopsis (Hpa) is a pathogenic microbe that survives on the living leaf tissue of Arabidopsis thaliana (Arabidopsis). When Arabidopsis is infected with Hpa it can develop downy mildew, a growth- and development-inhibiting disease. The Arabidopsis RPP7 gene encodes an immune receptor that can recongize certain Hpa races. In response to Hpa recognition RPP7 triggers localized death of Arabidopsis cells around the invading pathogen. This action removes resources from Hpa and also floods the infected tissues with plant metabolites toxic for the pathogen resulting in disease resistance. Expression of RPP7 is inducible upon pathogen recognition but needs to be very tightly controlled, as overexpression of this gene causes rampant cell death and can be lethal for the plant. Our lab is particularly interested in processes that regulate RPP7 gene expression. RPP7 is interrupted by a transposon. This causes the gene to create two alterative transcript isoforms, one encoding the complete protein and a smaller one without coding information. This alternative polyadenylation mechanism, is controlled by the epigenetic regulator EDM2. The EDM2 gene itself also produces alternative transcpit isoforms encoding different proteins. In preliminary studies, our lab has found that the ratios of these different transcript isoforms change in response to an Hpa infection. We are curious as to what function these different isoforms preform. Throughout this REU Program I plan to repeat and expand these preliminary studies in order to understand how these ratios are changing in response to an infection and if the predicted protein isoforms execute different functions.

  

Joshua Johnson, Central College (Borkovich lab)

 joahua

 Fluorescent Tagging of a Family of G-Protein Coupled Receptors Involved in Plant Cell Wall Degradation

G-protein coupled receptors (GPCRs) are membrane associated proteins that are important for regulating growth and sensing environmental stimuli in many organisms, including, fungi, plants, and humans.  The PTH11-like GPCRs are of interest because little is known about this class outside of the organism Magnaporthe oryzae, which is a pathogen of rice (Oryza sativa). The GPCR PTH-11 is necessary for host surface recognition and pathogenicity in M. oryzae. Better understanding of this protein can lead to advances in agriculture. In particular, greater knowledge of PTH11-like GPCRs could help increase yields of rice by targeting the pathogenicity of M. oryzae. The model filamentous fungus Neurospora crassa has 27 putative genes for PTH11-like GPCRs, which is of interest because N. crassa is responsible for the turnover of cellulose in the environment, rather than functioning as a plant pathogen. In addition, N. crassa has a fully sequenced genome and a full library of gene deletion mutants for each gene. These qualities make it an ideal model organism for study of the PTH11-like gene family. In this study, a green fluorescent protein tag was added to each of the putative genes, as well as a V5 tag, which is a small epitope with an easily accessible antibody. The vectors with the genes coding for tagged proteins were transformed into N. crassa, creating tagged versions of each PTH-11 like GPCR. The fluorescent GPCRs were used to study localization of the GPCR when growing on different carbon sources as well as co-localization with red fluorescent protein-tagged heterotrimeric Galpha proteins.

 

Vanessa Peterson,    California Polytechnic State University, San Luis Obispo; (Yang lab)

 vanessa

 Functional Role of Membrane Microdomains in Auxin-mediated Pavement Cell Formation

Membrane microdomains enriched in sphingolipids and sterols have been implicated in the functioning of a plethora of cellular signaling processes. The overarching goal of this project is to explore the functional role of membrane microdomains in the formation of Arabidopsis leaf epidermal pavement cells. The central hypothesis to be tested is that auxin regulates the dynamics of membrane microdomains at the plasma membrane, leading to the establishment of the lobe-indentation asymmetry. To test our hypothesis, we propose two main aims. Aim 1 will test whether the integrity of lipid microdomains is required for PC formation. The sterol-and-sphingolipid-deficient Arabidopsis mutants will be used for this part of the work. PC phenotype in these mutants will be visualized using confocal laser scanning microscopy. If our hypothesis is correct, we should observe defected PC formation in these mutants. Aim 2 will investigate how auxin regulates the specific recruitment of proteins into membrane microdomains. It has long been suggested that proteins involved in signal transduction can be selectively induced or excluded from the lipid microdomains during intracellular signaling. Biochemical approaches will be used for this part of the work. We anticipate an altered protein composition of membrane microdomains after auxin treatment. Uncovering the link between auxin signaling and membrane dynamics in PC formation will be an important advancement in plant developmental biology and cell signaling.

  

Tamera Taylor, Chaffey Community College (X. Chen lab)

 tamera

 Exploration of D-Body Formation in Arabidopsis

MicroRNAs (miRNA) are a type of small RNA, consisting of 20 to 40 nucleotides, associated with gene silencing in eukaryotes. They play an essential role in development and physiological regulation by RNA degradation or translation inhibition.  In Arabidopsis, primary transcripts are processed into miRNA/miRNA duplexes by Dicer-like 1 (DCL1) in the nucleus. DCL1 is colocalized with partner proteins HYL1 and SE in membraneless organelles called dicing bodies (d-bodies). It has been proposed in mammalian cells that membraneless organelles, like stress granules, are formed through a process of liquid-liquid phase separation (LLPS) that can be promoted by proteins containing intrinsically disordered regions (IDR). In Arabidopsis, HYL1 contains an IDR at its C-terminus, suggesting the possibility for it to undergo LLPS and form d-bodies in a comparable manner. To uncover the mechanism of d-body formation in Arabidopsis, we recombinantly express HYL1 fused with SNAP tag, then coupled with SNAP-Surface 488, to monitor the dynamic behavior of HYL1 in vitro and test its ability to undergo LLPS. We will characterize the roles of previously identified genes required in d-body formation in vitro by recombinantly expressing them and checking their influence on HYL1 and its phase separated droplet. These experiments will use techniques in PCR, agarose gel electrophoresis, DNA extraction, ligation and microscopy.

 

Ngoc (Iris) Thai Hieu Tran, Riverside City College (M. Chen lab)

 ngco

 Translocating mechanism of HEMERA from plastids to nucleus

For cells to survive and grow in different environments, communication between subcellular organelles is the key. Notably, various studies showed that there are many dual-localized proteins with multiple functions in plants, and a number of those enact the retrograde pathway from plastids to the nucleus. HEMERA (HMR) in Arabidopsis thaliana plays important roles in chloroplast biogenesis as well as light signaling in nucleus regarding hypocotyl growth. Although previous studies showed that HMR first goes to plastids via a transit peptide at the N-terminus, due to the predicted nuclear localization signals of HMR does not function as expected, the sorting mechanism of this protein from plastids to the nucleus is still poorly understood. Besides, the discovery of the protein RCB (Regulator for Chloroplast Biogenesis) suggested that this protein could interact directly with HMR, and together they affect the processes of light signaling in nucleus and chloroplast biogenesis. Therefore, to test the hypothesis that the interaction between HMR and RCB enhances the translocation of HMR into the nucleus, I will use Agrobacterium containing genes which encode for various versions of HMR fused with YFP and RCB fused with CFP, then expressing them separately and together in tobacco by infiltration. The result of each transformation will be observed under Fluorescent Microscope. The outcome of this experiment is expected to provide new insight into how HMR translocates from chloroplast to nucleus specifically, and the communication between plastids and nucleus in general.

 

Aeriel Vanluesauls , Riverside City College (Dehesh lab)

 aeriel

 PhosphoChloroplast Retrograde Signaling in Response to High Light Stress in Arabidopsis thaliana

The metabolite 2-C-methyl-D-ethyritol 2,4-cyclodiphosphate (MEcPP) is produced in the MEP pathway and accumulates in plants in response to external stress. Generally speaking, when a plant is exposed to a stress, it first senses the stress, activates a signaling pathway, and then initiates a response. Our experiment focuses on high light stress, which is sensed by the chloroplast. When high light is sensed, MEcPP accumulates triggering a signaling pathway from the chloroplast to the nucleus, ultimately regulating gene expression.  How this signal localizes to the nucleus is still unknown. Our hypothesis is that the accumulation of phosphoinositides in the nucleus regulates gene expression in response to high light. This response can be observed using a phosphoinositide sensor molecule and observing the sample by confocal microscopy. Additionally, we will compare the capabilities of triggering the retrograde response to high light conditions or MEcPP treatments in Col-0 wild type plants and various knockout mutants impaired in PI production. To isolate the homozygous mutants we will use PCR. Then, we will use quantitative RT-PCR to determine which plants are knockouts. Once we have selected the homozygous knockout mutants, we will expose them to the high light or MEcPP and use quantitative RT-PCR to determine if specific genes are activated by the retrograde response. If our hypothesis is correct we should find HPL and bzip60.

 

 James Jen Yen, UC Riverside (Nothnagel lab)

 james

 Solubilization and renaturation of plant cell wall methyltransferases expressed in E. coli

Research on plant cell walls has value towards increasing knowledge about plant structure and function and towards providing environmentally friendly energy for society. While combustion of petroleum deposits directly releases climate-changing carbon dioxide into the environment, production and combustion of biofuels derived from plant cell walls is nearly carbon neutral and thus environmentally advantageous. This project focuses on methyltransferases that add O-methyl ethers to sugars in plant cell walls. Two methyltransferases, MT1 and MT6, encoded by Physcomitrella patens genes cause synthesis of 3-O-methylgalactosyl residues when transgenically expressed in tobacco. This result is curious because the moss cell wall contains many 3-O-methylrhamnosyl residues but few, if any, 3-O-methylgalactosyl residues. The hypothesis is that the MT1 and MT6 methyltransferases can methylate either rhamnosyl or galactosyl residues, depending on substrate conditions. The approach is to separately express the methyltransferases in E. coli, purify the tagged proteins, and biochemically assay their enzymatic activities under controlled conditions. Physcomitrella cDNAs corresponding to MT1 and MT6 were modified to remove the transmembrane domain and to add a short N-terminal peptide that binds to modified streptavidin to enable affinity purification. This modified cDNA was cloned into the pASK-IBA16 plasmid under the control of the tetA promoter so expression is induced by anhydrotetracycline. The plasmid also adds a signal sequence targeting the periplasmic space, promising easier downstream purification. Protein gel electrophoresis indicates, however, that the transgenic polypeptides probably accumulate in inclusion bodies. Solubilization and renaturation of polypeptides from inclusion bodies are currently under investigation.


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