“Probing Nuclei in Insect and Plant Cells”

Project Summary:

The nucleus is an integral cellular structure that protects and organizes the eukaryotic genome. While the role of the nucleus has been well characterized, less is known about factors that can potentially impact features of nuclear architecture including size, shape, and stiffness; even less is known for non-mammalian organisms. Through two experiments, we have investigated 1) the correlation of genome size and repetitive DNA content to nuclear stiffness across three different insect species; and 2) the impact of disrupting the nuclear lamina (through mutations in NMCP genes) on nuclear size and shape in tomato.

To gauge how nuclear stiffness is affected by the size of the genome and the fraction of repetitive DNA it contains, we cultured cell lines from three different insect taxa and analyzed nuclear stiffness. Well-separated cells adhered to a glass surface were probed individually using a nanoindentation device mounted on an epifluorescence microscope. Our preliminary evidence suggests that a simple correlation does not exist between the genome size and nuclear stiffness of insects.

To study the role the plant nuclear lamina plays in governing nuclear architecture compared to the mammalian nuclear lamina, we raised tomato plants with mutations in the plant laminar protein-encoding NMCP genes to create three mutant genotypes whose nuclei could be analyzed. In doing so, we found that nuclei in mutant tomato guard and epithelial cells vary significantly from their wild-type counterparts, becoming more spherical in shape – a result different from that observed in comparable mammalian nuclei with nuclear lamina defects.

My Experience:

The time I spent as a summer intern in Dr. Eric Richards’ lab at BTI has been one of the most rewarding experiences of my academic life, maturing me not only as a researcher but as an individual. With Eric’s mentorship and the support of our research community, I applied my prior experiences in biophysics to contribute to three projects and developed new lab skills in cell biology and genetics.

In addition, I engaged in professional development events, from attending weekly research presentations to exploring different graduate programs through Cornell’s DGS event. Through these opportunities, I was exposed to various research fields and met with faculty to learn more about their career paths.

Outside of work, I found solace in connecting with other interns and experiencing the summer together. Although our time together was short, the friendships I have formed through BTI’s PGRP are ones that will span beyond Ithaca.

“Exploring the Characteristics of Plant Nuclei”

Project Summary:

While the structure of the nucleus is well-studied, we are interested in investigating the genomic characteristics that govern nuclear architecture and physical properties. This summer, we developed a protocol to prepare intact nuclei from tomato plants, and we also studied the changes in nuclear phenotypes caused by mutations in the tomato NMCP2 gene, which encodes a nuclear lamina protein.

The nucleus is an integral part of eukaryotic cells, as it contains, protects and organizes the genome. While the structure of the nucleus is well-studied, the genomic characteristics that govern the nuclear architecture and physical properties remain unclear. This summer, we developed a nuclear isolation method for future studies in nuclear biomechanics to probe how genome parameters affect nuclear properties. We developed a protocol to prepare intact nuclei from Solanum lycopersicum (tomato) plants on microscope slides and petri dishes with minimal debris. This procedure will facilitate further investigations on the mechanical properties of plant nuclei, and we can use this procedure to determine whether plant nuclei are pliable or rigid. We also studied the changes in nuclear phenotypes caused by mutations in the tomato NMCP2 gene, which encodes a nuclear lamina protein. In particular, we found that individuals homozygous for the nmcp2-3 hypomorphic allele had nuclei that were smaller and rounder than in wild-type individuals. Furthermore, we found some evidence for nuclear positional differences between wild-type individuals and nmcp2-3 mutants. In nmcp2-3 mutants, the nuclei in the two guard cells of each stomata tended to form a more drastic angle with the length of the stomata. This larger offset angle could be correlated to the smaller and rounder shape of the nuclei, and/or abnormal interactions between the nucleus and the cytoskeleton. Our discovery of a hypomorphic but viable nmcp2-3 allele (nmcp2 null alleles are lethal) will allow us to include study of the NMCP2 gene in our future investigation of nuclear biomechanics.

My Experience:

As an intern at BTI this summer, I gained experience in lab techniques including PCR and fluorescence microscopy. I also learned about general research practices, such as maintaining a good lab notebook and designing experiments. Going into the program, I had no previous research experience and was quite nervous about being in a lab environment, and I often worried that I was not cut out for biology research. However, with the support of my mentor and fellow interns, I quickly gained confidence and became comfortable with asking questions whenever I needed guidance. In addition, my graduate school mentors provided me with valuable advice on how to prepare for a career in scientific research. This program helped me hone the skills I have learned in previous biology coursework and apply my knowledge to real-life experiments, all while advancing my technical knowledge and allowing me to further explore different career options.

Genomic Instability of Duplicated Disease Resistance genes in Arabidopsis thaliana

Project Summary

SNC1 is a pathogen resistance (R-) gene in Arabidopisis thaliana. SNC1 duplication leads to the dwarfed mutant classified as bal, whose most notable characteristic is its phenotypic instability following mutagenesis. This mutagen-induced phenotypic instability of the bal mutants was first noted after a genetic suppressor screen in which a statically significant number of phenotypically “normal” plants were recovered; these reversion or suppression events were named bal à BAL*, and subsequent molecular analysis indicated that one SNC1 copy is inactivated in BAL* plants.

Two different models were formulated to explain the high frequency recovery of BAL* plants. The first hypothesis posits that hypermutation of SNC1 occurs due to local stress-induced mutagenesis, as a consequence of DNA damage. This model stands on the idea that as DNA damage occurs it leads to localized mutagenesis through induction of low-fidelity DNA polymerases, and implies that the mutation rate of the SNC1 gene in the bal variant is elevated preferentially. The second hypothesis is that selection within the meristem causes the high frequency recovery of BAL* phenotypes in the progeny.  It is reasoned that SNC1 overexpression results in a decrease in fitness, establishing competition among bal and BAL* stem cells. Stem cells carrying a BAL* allele would have a growth advantage and as a result be more likely to contribute to the subsequent formation of reproductive tissues.

These models can be distinguished by observing the types of SNC1 mutations that occur in response to different mutagenic treatments.  Previous experiments indicated the EMS treatment of bal plants leads to C/G à T/A mutations in SNC1. My project looked at the types of snc1 mutations that were recovered in BAL* plants generated by fast neutron mutagenesis, which causes deletions and DNA rearrangements versus base-pair changes. To characterize the mutation spectrum, end-point and quantitative PCR was used in addition to Southern blots and Sanger sequencing. The results of my study support the meristem selection model since the mutations that I characterized reflect the types of mutations expected from fast neutron mutagenesis.

My Experience

The Boyce Thompson Institute internship has provided me with invaluable insight into some of the rigorous and exciting work conducted in a professional research environment. Through the excellent guidance of my mentor, Erika Hughes, I gained a complete understanding of numerous lab techniques, as well as confidence in my own abilities conducting research. While working in the Richards lab I was exposed to a wide range of new research topics, specifically in genetics, seeking to answer fundamental questions in biology. Which has aided me in seeing more clearly the true importance of research in the plant sciences. This internship has also been a good preview for my future, as I plan to matriculate into graduate school to continue my study of biology.

Testing the Role of Error-Prone DNA Polymerases in Genetic Instability of Gene Duplications

Project Summary

Organisms like Arabidopsis thaliana activate error-prone DNA polymerases when under extreme stress, such as conditions causing high damage to DNA. We used DNA alkylating agent, ethylmethane sulfonate (EMS), to mutagenize a gene duplication mutant of Arabidopsis, called bal. This duplication causes a dwarfed phenotype involving shrunken rosettes. Mutants are stable under natural conditions, but after treating mutant seeds with EMS, a high frequency of progeny show bal phenotype suppression. This project was designed to determine whether error-prone DNA polymerases are involved in the high number of suppressed progeny. My experiments involved planting mutagenized and non-mutagenized seeds of various Arabidopsis genotypes: bal control and bal/error-prone DNA polymerase double mutants. Adult plants were characterized based on number of individuals containing suppressed phenotypes (e.g., expanded, flat leaves; tall flowering stems). Effectiveness of EMS mutagenesis was evaluated by opening Arabidopsis siliques to score plants giving rise to aborted seeds.  This data confirmed mutagenesis induced lethal mutations in treated populations unlike mock-treated lines. As expected, EMS treatment produced a higher return in plants suppressing the balphenotype compared to seeds without mutagenesis. However, the difference in the return of the suppressed phenotype, between mutagenized seeds of bal control and bal/error-prone DNA polymerase double mutants, was not significant. These findings suggest these error-prone DNA polymerases are not involved in the high rate of recovery of suppressed bal phenotype plants after EMS mutagenesis. These error-prone polymerases may act redundantly, and it will be important to compare sector frequencies in further error-prone polymerase mutants.

My Experience

Being at Boyce Thompson Institute has set the bar high for future internships I will be involved in. This was my first summer research experience, and I have learned so much by being immersed in this community. It has conceptually been a great help to handle protocols and lab procedures that were once just words read in a textbook, words mentioned by my professors, or material that was not even a part of my curriculum. With the tremendous support of the members of the Richards lab, I have been making a transition in my way of thinking. Rather than just taking in information, they have helped me to apply it, and I have enjoyed the process. This experience has also given me a better understanding of paths my life can take with opportunities I now know are available. I am excited more than ever about what my future holds.

Phenotypic and genetic instability of a gene duplication in Arabidopsis thaliana

Project Summary

A duplication of the pathogen Resistance gene SNC1 in Arabidopsis thaliana results in a semi-dominant, curly leaved, dwarfing phenotype called bal.  A 55-kb duplication of the R-gene cluster containing SNC1 was recovered from a ddmt (DNA methylation) mutant background.  The Richards lab has been studying both the formation of the duplication and the unstable bal variant.  Overtime, mutagenized bal plants frequently produce chimeric offspring, called BAL*, that appear part wild-type and part bal.  Previous work from Dr. Richard’s lab using the mutagen ethyl methanesulfonate (EMS) indicates that mutations can inactivate one of the duplicated SNC1 genes, resulting in the chimeric/BAL* phenotype.  The rate of mutation, thus the number of BAL* individuals, is elevated above the expected frequency in mutagenized populations.  My project was to investigate if fast neutron mutagenesis could also induce mutations that inactivate SNC1.  Furthermore, if these mutations occur, where do they occur and are they stably inherited?  To help answer these questions, I employed fast-neutron mutagenesis to generate new BAL* plants.  I sequenced the SNC1 gene in these plants with Polymerase Chain Reaction (PCR) and Sanger sequencing techniques.  This work will likely contribute in the understanding of DNA repair in response to DNA damage.

My Experience

My time with the Richards Lab and the Plant Genome Research Program enabled me  to explore research in plant biology and genetics.  It has been a pleasure to get to know members of the lab and other interns interested in research.  I also enjoyed the chance to learn about many different areas of science through seminars, training, and events.  This internship has encouraged me to pursue graduate studies in plant research after I complete my bachelors degree in biology.

A cross species exploration of nuclear morphology

Our knowledge and understanding of the structures and mechanisms responsible for nuclear organization are based primarily on research in animal cells. In order to address the lack of understanding of plant nuclear morphology, new research specifically targeting the nuclear organization of plants has begun. However, much of this research is limited to the popular model plant, Arabidopsis thaliana. In order to broaden the base of research in this area, my project included not only A. thaliana, but three other plant species: Medicago truncatula, Nicotiana benthamiana and Solanum lycopersicum. The main objectives of the project were to determine whether there are any differences in the nuclear morphology among these plant species in regards to cell/organ-specificity in nuclear size and shape. In addition, we investigated whether environmental conditions could affect nuclear shape, using a transgenic A. thaliana line carrying a nuclear-localized GFP marker. Specifically, we exposed this line to different osmotic environments and observed whether any changes in the nuclear shape or size resulted. From this project, we have shown that differentiated (non-spherical) nuclear shapes are seen in Medicago, Nicotiana benthamiana and Solanum lycopersicum root tips, roots, hypocotyls, leaves and anthers. We also observed that altering osmotic conditions of A. thaliana does have a visual effect on the nuclear shape and size of the nuclei, suggesting that nuclear structure is easily perturbed by this type of environmental stress.

My Experience

I have always been an individual who enjoys going outside and exploring the world around me. Ever since I was old enough to go on hikes and adventures down by the Redwood River in Minnesota with my dad, I have wanted to have a job where I could study plants in and out of their natural habitat. As a PGRP intern at BTI, I was given the opportunity to dive deep into the area of plant biology at a molecular level and acquire new skills and techniques that I will carry out throughout my scientific career. The Richards lab gave me an opportunity to experience a research team that was enthusiastic and fun while still being serious about their work. I also gained confidence in my ability to think and work independently, yet felt comfortable seeking out new ideas and thoughts from my colleagues. I have decided that my career goal is to obtain a Biology Major, acquire a forestry position then transition to graduate school, and earn my PhD in Plant Ecology/Horticulture.

Epigenetic Regulation of Sadhu Transposable Elements in Arabidopsis thaliana

Studies exploring natural variation of the Arabidopsis transcriptome uncovered a family of retrotransponsons, dubbed Sadhu elements, which show genetic and epigenetic variation among different accessions. Several of these elements have been further characterized and appear to be meiotically stable “epialleles,” epigenetic alleles that differ only in cytosine methylation state and thus expression state. In this study, the knowledge of epigenetic variation in Sadhu elements was further explored by investigating potential developmental regulation of epigenetic silencing. Both endpoint and quantitative RT-PCR were used to determine whether the epigenetic state of Sadhu elements vary between tissue types in two standard wild-type strains: the Col and Ler accessions. In addition, public datasets in the Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra), a depository for next-generation sequencing data, were mined to determine whether various abiotic stresses affected the expression of Sadhu elements and to confirm tissue expression findings. Many retrotransposons are reputedly under epigenetic regulation and have been implicated in playing important roles in stress adaptation and evolution. Our study contributes to knowledge of both retrotransposon regulation and epiallele stability by investigating the expression of Sadhu elements in different tissues and under stress.

My Experience

As part of the PGRP program, I was provided with the opportunity to work on cutting edge plant biology research in an unintimidating, welcoming environment. Weekly seminars provided by the program were both interesting and informative. In addition to lab related activities, plenty of opportunities were available to explore the Ithaca community. Overall, my summer at BTI has cemented my interest in research and has instilled me with confidence as I move forward towards graduate school.

Natural Epigenetic Variation in Sadhu Retroposons in Arabidopsis

The Arabidopsis genome contains a family of retroposons that are called Sadhu elements. One of these elements, Sadhu1-1, was previously shown to exhibit epigenetic variation with respect to DNA methylation among different wild-type accessions of Arabidopsis. Further, this variation was not strictly controlled by genetic variation acting in trans or by nucleotide polymorphisms at the locus, suggesting that this epigenetic variation was an example of an epiallele. It was also demonstrated that an inverse correlation exists between DNA methylation and expression of the Sadhu1-1 element. My project extended these findings by investigating the relationship between DNA methylation and nucleotide sequence variation at a second Sadhu element, Sadhu6-1. We found multiple strains that shared identical sequence at Sadhu6-1 but had varying methylation patterns. This finding demonstrates a situation where an epiallele exists at a retroposon that is not strictly determined by the underlying nucleotide sequence. We also found a negative correlation between DNA methylation and expression at Sadhu6-1, as was previously reported for Sadhu1-1. These findings suggest that DNA methylation is a regulating mechanism at Sadhu retroposons.

My Experience

I’m not entirely sure what I want to do after my senior year of college but even before my summer internship at BTI, I was already pretty sure that I wanted to be involved in scientific research. This experience was reassuring. Being given the opportunity to work on my own project with the guidance of a mentor, working at BTI gave me research experience that will be very valuable in preparing me for the future. Overall I had a wonderful experience working at the Boyce Thompson Institute. It is a great place to do research, my fellow interns were a joy to work with, and Ithaca is an awesome place to be.

Functional dissection of the VIM1 protein and its role in cytosine methylation in Arabidopsis

Epigenetics is the study of chromatin modifications, such as cytosine methylation and histone modification, which affect gene expression. VARIANT IN METHYLATION 1 (VIM1) is an epigenetic regulator required for both maintenance of DNA methylation and centromeric heterochromatin compaction in Arabidopsis thaliana. The VIM1 protein has a methyl DNA binding SRA (SET- and RING-associated) domain, as well as a PHD(plant homeo-domain) and two RING (presumptive ubiquitin E3 ligase) domains. We hypothesized that the VIM1 protein SRA domain is involved in maintaining cytosine methylation in Arabidopsis but that the RING and PHD domains are dispensable. To test this hypothesis, PHD and RING point mutants were expressed in vim1 null mutants. We studied whether or not the centromeric repeats were fully methylated in transgenic plants expressing the mutated versions of VIM1 using DNA blots of genomic DNA digested with methylation-sensitive endonucleases. To test transgene expression of our point mutants, we looked at mRNA expression of VIM1 using RT-PCR. To supplement our experimental work on VIM1 domains, we studied natural variation of these domains using protein sequence alignments generated from the Arabidopsis 1001 genome database (http://signal.salk.edu/atg1001/3.0/gebrowser.php). Our research can lead to new knowledge about epigenetic mechanisms in plants. Furthermore, our research has implications for humans because VIM1 has a human homolog, UHRF1.

My Experience

As a PGRP intern at BTI, I was given the opportunity to learn more about plant biology on a molecular basis and practice new research techniques. The environment was very opened and supportive. Everybody in the Richards’ lab was very enthusiastic and welcoming. I know the skills and knowledge I gained over the summer will help me in future endeavors. The program exposed me to many different aspects of plant research separate from my project and gave me an accurate picture of what plant biology research entailed. Outside of lab, Ithaca is a great place to be over the summer. I have made strong connections and friends over the summer and I’m grateful for the opportunities the PGRP internship has given me.

Investigating Natural Variation of Epigenetic Regulation of the Promoter of the Stress Response Gene ESM1

Epithiospecifier Modifier 1 (ESM1) is a stress response gene affecting secondary metabolite formation in response to herbivory. In the promoter region of this gene is a series of repetitive elements, which resemble those in other areas of the genome that have previously been shown to be epigenetically controlled. This led to the hypothesis that the promoter region of ESM1 might be under epigenetic control of some type. I investigated the possibility of epigenetic control mechanisms affecting the promoter region of ESM1 and inducing differential expression of the gene itself, looking for a correlation between the two variables. I focused specifically on DNA methylation in several different strains of Arabidopsis thaliana, using techniques such as quantitative real time PCR, restriction enzyme digests, and sequencing to gain a better understanding of how epigenetic factors interact with this locus.

The Missing LINC: The Relationship Between Nuclear Structure and Epigenetic Regulation

The Arabidopsis LINC proteins are nuclear coiled-coil proteins suspected to be analogs of lamin proteins, which compose the nuclear lamina in animal cells. A mutation in the LINC1 gene causes small, round nuclei rather than the normal, rod or spindle shaped nuclei, leading to the hypothesis that mutation of this gene might also have great effects on nuclear organization of the plant cell. For example, this compacting of the nucleus has been shown to decrease the number of chromocenters, regions of dense DNA packaging where centromeres and other repetitive DNA are stored in interphase. However, it is unknown whether the smaller space within a linc1 nucleus affects epigenetic regulation, the activation and silencing of genes without nucleotide sequence changes. I tested several types of DNA sequences found in chromocenters for either a decrease in cytosine methylation, signaling a loss of heterochromatin, or an increase indicating compaction of existing heterochromatin. The evidence of my experiments to date has been inconclusive, showing little to no qualitative shifts in cytosine methylation in linc mutants. The elucidation of the effects of linc mutations could lead to a greater understanding of eukaryotic nuclear structure in general, allowing for greater knowledge in fields even as far-reaching as animal systems.