Know when to hold ‘em: Folate Biofortification via Stabilization and Storage

Folates are B-vitamins essential for creating energy-rich molecules like ATP, and for regulating genetic repair and expression. Because humans do not produce folate, maintaining a folate-rich diet is crucial, especially for women of childbearing age, because deficiency can lead to devastating neural tube defects in developing fetuses. Other afflictions include megaloblastic anemia and several cancers. Folate deficiency is seen across the world, and although many countries supplement their cereals with the synthetic folate derivative folic acid, developing countries where folate deficiency is common often don’t have these resources. Not only that, folic acid has shortcomings even in developed countries, not the least of which is the potential carcinogenicity risk of high folic acid intake. There is growing research on biofortifying plants to provide a higher supply of natural folates, but progress has been slow, in part because folates are highly unstable and prone to degradation. Even if more folates are produced in the plant, they may not remain until consumption. Therefore, the aim of this work is to understand the role of Folate Binding Protein on folate stabilization through in planta and in vitro methods, to support the creation of tomato plants with more bioavailable folates.

I am delighted to be a part of the 2024 BTI REU cohort! Being surrounded by amazing plant science this summer has been even more wonderful than I expected, and opened exciting new doors for me in every part of my science journey. Particularly fascinating was the chance to work in a USDA lab and get a feel for what governmental research is like, but learning how to use LC-MS, engaging in science communication with ScienceIRL, and visiting the CHESS labs were also formative experiences for me. I’m deeply grateful to my mentor John Ramsey, and also to my PIs Jim Giovannoni and Ted Thannhauser. All opened their doors to me, made me feel welcomed, and went above and beyond to help me reach my professional goals. Now that I am entering my final year in my undergraduate degree, I am looking forward to beginning my PhD and fellowship grant applications, and I am so appreciative to have an amazing set of experiences and such a bright community to guide me.

High-throughput phenotyping of Galápagos Island tomatoes with computer vision

Cultivated tomato plants and their wild relatives exhibit considerable phenotypic diversity and are therefore a useful model system in fruit biology. To measure quantitative trait variation in Solanum cheesmaniae, a wild tomato relative native to the Galápagos Islands, we developed a computer vision-based approach. The fruit are first photographed along with a color checker and photomacrographic scale. During image processing, the fruit are segmented from the image background, and size, shape, and color measurements are recorded. We demonstrate the effectiveness of our tool by comparing the computational measurements with those taken manually. Fruit characteristics across 25 accessions of S. cheesmaniae were analyzed to identify phylogenetic and geographic patterns. Future work will apply our pipeline to characterize a diversity panel that spans wide genotypic and phenotypic variation in wild and cultivated tomato species. We aim to use computationally-measured phenotypes to predict physiological or biochemical traits, thus saving time and resources while increasing the consistency and accuracy of plant phenotype analysis. The ability to easily and consistently measure agronomically important plant phenotypes via computer vision will assist plant breeders and geneticists in improving crop varieties for commercial production.

During my time in BTI’s REU program, I gained a new appreciation for how plants serve as model systems for the development of computational biology tools. By working in the field, greenhouse, and lab, I was able to participate in each component of the project and understand the biological basis for the computational tool I was creating. I enjoyed immersing myself in the work, while also taking time for activities such as hiking, blueberry picking, and shopping at the Ithaca Farmers Market. The computational skills and laboratory techniques that I learned this summer are transferable to any research or data analysis project. My experience at BTI strengthened my interest in computational biology and inspired me to keep exploring the many opportunities in genomics and plant research.

Carotenoid analysis of gene-edited rin mutant tomatoes

Tomato (Solanum lycopersicum) serves as a model system to study ripening in fleshy fruit due to its fast rate of growth (3-4 months) and ease of genetic transformation. The rin (ripening inhibitor) mutation is naturally occurring in tomato, and results in a fused chimeric message between the RIN gene (transcription factor known to regulate ripening processes) and the adjacent MC gene (transcription factor involved in the development of plant inflorescence) on the chromosome. In this fused message, the 8th exon in RIN is deleted and part of the promoter in MC is deleted. The result of this mutation is fruit that fails to produce ethylene (a ripening hormone), never synthesizes carotenoids (responsible for red/orange/yellow color), doesn’t soften, and doesn’t develop a ripe aroma. To determine why the rin mutant tomatoes never ripen, CRISPR Cas-9 was used to effectively knock different parts of the RIN/MC message, and observe the phenotypes of the CRISPR lines. Carotenoid levels were measured in each CRISPR line to find the difference between carotenoid content in CRISPR lines compared to wild type tomatoes as well as the rin mutant, with the goal of discovering why the mutant tomato doesn’t ripen. With this knowledge, tomatoes that ripen slower, ship easier, and have a longer shelf life, can be used to feed the world.

I have had a great experience in the Giovannoni lab this summer. I have improved my ability to run PCR and gels, and learned new skills like gel purification, DNA extraction, and carotenoid extraction. I really enjoyed attending the weekly presentations and symposiums, and working in a hands-on lab environment. I also enjoyed getting to know my fellow high school interns, as well as the undergraduate interns. I would like to thank Jim Giovannoni for giving me the opportunity to work in his lab this summer. I also especially want to thank my mentor, Julia Vrebalov, for answering endless questions, guiding me through my research, and being patient with me and my fellow interns throughout the learning experience.

Exploring the macrolous rin mutation

The tomato (Solanum lycopersicum) has served as the model system to understand ripening processes because it is characterized well genetically, grows quickly (typically 3-4 months), and is of great economic importance. The RIN gene in wild type (WT) tomatoes is a transcription factor and an important component of the ripening process. However, the naturally occurring rin mutant makes all ripening processes impaired including ethylene production, (a hormone that triggers fruit ripening), carotenoid biosynthesis, (development of red pigment in the fruit) in addition to normal changes in the color, texture, and flavor. A section of the C domain in the MC gene is the putative EAR (ethylene-responsive element binding factor-associated amphiphilic repression) domain. This region was previously suggested to act as a repressor and cause the severe non-ripening phenotype. In order to determine which region of the MC gene acts as a repressor, CRISPR-Cas9 was used to target the MC-EAR domain as well as the whole MC part of the gene in the rin mutant.

Working in the Giovannoni Lab this summer has been an incredibly enriching experience. I was exposed to what a scientific career may look like and gained a better understanding of the research process. Working in a formal lab setting allowed me to cultivate crucial research skills and greatly improved my lab technique. Overall, this internship was an incredibly positive experience where I was able to better understand my interests and passions, as well as make great connections. I am incredibly grateful to my mentor, Julia Vrebalov, and to BTI for giving me this wonderful opportunity.

Project Summary:

Commercial tomato accessions have been bred for specific traits that allow for ease of harvest and marketability, but many lack important nutrients and flavor compounds. The focus of this study is to identify variation in carotenoid metabolite accumulation and gene expression for different tomato accessions and to investigate correlations between the two phenotypes. Carotenoid extractions were performed for three S. lycopersicum var. cerasiforme accessions, which are cherry tomatoes, and five S. lycopersicum var. lycopersicum accessions, which include both heirloom and modern accessions. Gene expression data was already available for these accessions. RNA extractions were performed on additional accessions to obtain a larger set of gene expression data for future studies. Among the additional accessions are several wild species, which were also included in the carotenoid accumulation analysis. However, since gene expression data was not yet available, those accessions could not be included in the correlation analysis. Data analyses were performed in R to obtain correlation values as well as the carotenoid and gene expression data. The correlation analyses indicated wide variation not only between the species analyzed, but also between accessions within the same type of species, which was unexpected. Variation was further analyzed between different fruit stages of the same accession. Potential future application of these findings includes use in plant breeding for the improvement of commercial tomato accessions to have higher nutrient density and flavor.

My Experience:

I had many good experiences and gained valuable research skills this summer! I really liked my lab and my project wet my whistle for plant breeding. Because of my project, I gained a greater appreciation for the variation and complexity within the tomato species. I’m also interested in graduate studies at Cornell and appreciated the opportunity to learn more about SIPS while I was here. I felt the DGS event was informative and found it helpful to talk with faculty one-on-one. Looking back on it, I feel an important aspect that made this experience great was the people. I really appreciate my mentor and her willingness to teach me and take time to answer my questions. Her efforts, as well as that of others, had a profound impact that allowed me to benefit from this experience academically, professionally, and personally.

“Exploring the Role of Ethylene in Transcriptional Regulation of the Melon Carotenoid Pathway”

Project Summary:

Ethylene is a phytohormone emitted by certain fleshy fruits and a key regulator of ripening in crops such as tomato, melon, banana, and pear. Ethylene’s role has been particularly well defined in the ripening process of tomato. In addition to the release of ethylene, an important parameter of ripening is the evolution of fruit color. In particular, carotenoid pigments are compounds integral to plant development and fruit quality, mediating both flavor and aroma and fulfilling roles as dietary antioxidants that lower blood pressure and help prevent heart disease (Lewinsohn et al 2005). In tomato, ethylene upregulates the expression of ripening-related transcription factors which collectively regulate the biosynthesis of carotenoids (Li et al 2019). While tomato offers a model organism tractable to genetic analysis, many fruit crops display variation across ethylene-dependent ripening systems. An example of this variation is evident in melon, where different varieties exhibit a range of climacteric and non-climacteric phenotypes. Accordingly, the role of ethylene in regulating the ripening process of melon is an area under active investigation, as the operation of the melon carotenoid pathway during ripening has only been broadly characterized compared to tomato, where the regulation of this pathway has been described in greater mechanistic detail. In particular, it remains uncertain as to why ethylene emissions and fruit carotenoids appear to operate independently during melon ripening (Guis 1997), whereas both processes are coupled in tomato. Our research hopes to clarify the applicability of ripening discoveries in tomato and further explore carotenoid biosynthesis in melon.

My Experience:

This internship gave me an opportunity that is hard to come by as a high schooler. I was able to work in a lab environment, learn about experimental design, and work with experienced scientists every day. I especially valued the nature of the mentor-mentee experience where I could ask questions about the smallest details of a procedure and expect support and guidance. I would also like to recognize the collaborative educational atmosphere at BTI. From weekly seminars to casual conversations with other interns and members of faculty outside of my lab, there was always an opportunity for learning. This experience has greatly expanded my knowledge of plant biology and shown me, in part, how scientific research is conducted.

“Identifying the Genetic Loci Contributing to Folate Accumulation in Tomato”

Project Summary:

Folates are one of the most important vitamins found in food. They serve as co-factors in the synthesis of DNA, methionine, and amino acids. While plants and microorganisms can synthesize folates, humans cannot. A deficiency in folate can lead to adverse health problems such as cardiovascular disease, anemia, dementia, and neural tube birth defects. Leafy greens are main contributors of folate, while most fruits are poor sources. Many countries fortify foods with synthetic folic acid. However, too much synthetic folic acid can be detrimental and cause health risks. Folate biofortification is an alternative way to increase natural folate in crops and diversify dietary sources of folate. This project focused on understanding the basis of folate accumulation in tomatoes, an economically and nutritionally important fruit. The Solanum pennellii introgression lines, which contain a genetically mapped, single introgression from S. pennellii in the genetic background of the domesticated tomato cv. M82. were previously accessed for genetic variation of folate content, and the expression of folate biosynthetic genes. Lines were chosen based on this information for pyramiding, to find crosses that resulted in higher folate and elucidate the causal gene(s) for the increased folate. Fruit folate levels were measured in two F3 populations using a microbiological assay. The results showed variation in folate content and were correlated with prior genotyping data. This also showed folate accumulation as a complex trait.  F2 lines, derived from a backcross between M82 and lines showing significant amounts of folate, were genotyped with genetic markers, and grown in the field. This will be a great resource for fine mapping the gene for folate accumulation and will be useful in the folate biofortification effort.

My Experience:

This program allowed me to understand and gain experience in a lab setting. Getting to meet and work with my mentor helped me really grapple with the idea of potentially going into research for graduate programs or possible future careers. Over the course of this summer, I have been able to be more confident and comfortable with conducting my own research. Getting to work with the many people at Boyce Thompson and the Giovannoni lab was an amazing experience because they were able to help me go in a direction that could benefit me in the long run. This program helps you understand the importance of plant biology and the many available career choices. It is one of the biggest research fields to work in and this program does a great job at describing it. After this program I plan on working in the plant biology field to gain more experience and find my way into areas that I would enjoy working long term.

“Pipeline development for identification of high-impact structural variants”

Project Summary:

Structural variants (SV) are recognized as a prominent source of genetic variation, with single variants manifesting significant influence over phenotypic expression, but their effects remain poorly understood due to computational limitations and a continued focus on SNPs. Addressing this need, we introduce the Variant Identification Pipeline with Expression Realization (VIPER), a robust computational pipeline capable of identifying and annotating structural variants with significant expressional impact. Using a publicly available structural variant library as reference (Alonge et al., 2020), VIPER accepts processed RNA sequence data and constructs a list of SV-gene pairs based on chromosomal position. These SV-gene pairs are then evaluated by their differential expression values, with pairs expressing significantly concatenated to form a library of high-impact SV-gene pairs. This library of causal genomic variants within tomato accessions, annotated for gene function, allowed for the identification of 105 high-impact leaf development variants, and the isolation of a single insertion with substantial effect on leaflet morphology.

My Experience:

In my time at BTI I always enjoyed a strong sense of community. I felt welcomed by every member of my lab, not only academically, even though everyone was happy to instruct me on new practices like seed germination in medium or tomato cultivation in the greenhouses, but socially. I was quickly welcomed into lab social activities like ice cream trips, hiking or festivals on the weekend, and group exercises on BTI’s lawn after a long day. Professionally, I was constantly offered new experiences or conversations with knowledgeable mentors who provided great advice on not only academic pursuits, but on how to apply to graduate studies or market myself appropriately. Outside of BTI, Ithaca manages to be welcoming to newcomers while retaining a wonderful closeness, and I quickly found social groups for rock climbing, sand volleyball and pickup basketball. I strongly recommend this REU for wonderful social and academic experiences.

“Metabolite Extractions from Tomato Fruit for Nutrient Composition and Gene Expression Analyses”

Project Summary:

Tomatoes are the world’s most important crop plant. According to the Food and Agriculture Organization, in 2019, 181 million tons of tomatoes were produced globally. Tomatoes have a relatively small genome and there is a great deal of information available about the genetic sequences of tomatoes. Tomatoes also have a relatively short generation time and are self-fertilizing with a homozygous genome, which makes it easy to study genetically identical plants and propagate them by seeds. All of these components make tomatoes an ideal model species. The major class of pigments underlying coloration in tomato fruit are carotenoids. Various fruit color phenotypes of tomatoes indicate different carotenoid profiles with contrasting levels of pigments, and this indicates different levels of carotenoid biosynthetic enzyme activity. The activity of these enzymes is what defines the accumulation of specific carotenoids, which results in the distinctive colors of tomatoes. Carotenoid biosynthesis genes are regulated at a transcriptional level. In order to determine the different accumulations of carotenoids, liquid chromatography is used to assess carotenoid profiles. These profiles can provide insight into the underlying genetic expression leading to differing accumulations of carotenoids in tomatoes. Once the carotenoid profiles are available, quantitative PCR can be performed to assess the expression levels of selected carotenoid biosynthesis genes. This will give us insight into which genes are responsible for the accumulation of certain carotenoids. With this information available, we have more knowledge about the genetic regulation of nutritious carotenoid profiles, which can be used to target the breeding of nutritious tomatoes.

My Experience:

Having this opportunity, to work in a lab and learn more about scientific research and plant biology, has been extremely helpful in giving me an idea of what I want to study and pursue in college. I really enjoyed working with a mentor and learning from her, whether it was basic yet complicated concepts that I’m not yet sure, I completely understand or small tricks to make lab work more precise and efficient, I really appreciated this experience. I was amazed by the vast amount of information that was available to me every day from the weekly lectures to seminars to overhearing conversations between people in the lab to the posters that are in every hallway. This internship has furthered my interest in scientific research and has given me much more insight into how scientific research is conducted and especially the importance of research in plant biology.

Development of gene constructs targeting fruit ripening transcription factors of previously characterized by incomplete repression.

With an increasing world population, challenges arise in producing and distributing enough food to feed the rising numbers of hungry mouths. One such challenge is maintaining proper nutrition where there is a lack of food. The time it takes to deliver produce to such areas may not be sufficient to ensure that it is fresh, or simply not be profitable with the given restraints of shelf life. One potential contribution to aid the resolution of this problem is understanding the function of genes involved in flower and fruit development, such as the MADS box genes. These genes are highly conserved transcription factors involved in plant organ development. Their role in flower and fruit development was best explained in Arabidopsis that have dry siliques as opposed to fleshy fruit. Identifying MADS box genes involved in fleshy fruit development and ripening, may lead to insights affecting the ripening process of fruits such as tomatoes.
The goal was to knock out potential MADS box tomato orthologs and define their role in fleshy fruit development and ripening. CRISPR-Cas9 is a novel technology developed from a bacterial defense mechanism and modified for gene silencing in eukaryotes. We anticipated to create CRISPR lines for the four candidate genes by designing constructs that contain two or three guide RNAs. The RNA guides will direct the Cas9 protein to the specific genomic region to generate a double stranded DNA break leading to the silencing of the gene function. Moving forward, the Giovannoni lab can use the constructs to observe the individual and combined knockout phenotypes to evaluate the gene functions and compare with previous RNAi phenotypes.

My Experience

My PGRP internship experience has been very eventful. I really appreciated all the exposure to different kinds of research going on at the weekly seminars. Also, digging into my research topic, learning how to write a proposal and present a poster were all new experiences that will help me later on. While writing, I was forced to consider the connection between my research and potential impacts on both agriculture and human health, which was something I never directly looked at before. The bioinformatics courses also provided an intriguing introduction to Unix and R, both of which I will come across again in furthering my education. Learning how to use programs to identify potential guides and align sequences was also something I had been hoping to learn. Overall, I am so grateful to have been here this summer!

Analysis of fruit-specific SLDML2 overexpression on ripening-related DNA demethylation in tomato fruit

Global agriculture today is facing the daunting problem of feeding a growing population on a decreasing amount of arable land. While increasing yields represents one strategy to address this issue, another is to reduce the spoilage of food that is already being produced. Fleshy fruits like tomato represent an attractive target for this improvement, as they are nutritionally-rich, but prone to spoilage upon ripening. Thus, the development of approaches to control the progression of ripening can be used to reduce food waste. My project focuses on the initiation of ripening in tomato fruit, with the goal of understanding the tight genetic controls underlying this process. Ripening in tomato is regulated by three interacting systems consisting of transcription factors, ethylene production, and DNA demethylation. I specifically focused on the role of DNA demethylation during ripening, as well as the gene DEMETER-LIKE DNA DEMETHYLASE 2 (SlDML2) that controls this process. I conducted my project using transgenic plants where expression of the SlDML2 gene occurs much earlier in fruit development than it would normally, and I characterized these lines using a combination of DNA methylation and expression analyses. I extracted DNA from two different fruit tissues, and used bisulfite conversion and sequencing to evaluate the differences in methylation levels between them. I also performed real-time polymerase chain reactions (qPCR) to measure the expression level of SlDML2 in different fruit tissues. I then evaluated this methylation and expression data while also considering observed fruit phenotypes to infer the role that SlDML2 and subsequent demethylation plays in ripening initiation. Ultimately, furthering this type of knowledge can lead to significant improvements in our ability to control the ripening process in a commercial setting and contribute to a more sustainable and less wasteful global food system.

My Experience

My experience at the Boyce Thompson Institute this summer has been amazing to say the least! Through the program I have been able to learn countless new laboratory techniques, gain insight in applying to graduate school and NSF funding opportunities, learn about an array of ongoing research in plant science, as well as be surrounded by a fun and encouraging group of people. My research specifically allowed me to delve into my interest in epigenetics and learn about the role it plays in tomato ripening. My mentor, Dan Evanich, was a phenomenal teacher and role model, and helped me understand and become confident in using techniques such as bisulfite conversion, demethylation analysis, qPCR, and DNA extraction. I also learned that research comes with many challenges, such as changing or restarting experiments when they don’t go as well as hoped, or hurriedly moving samples out of a freezer when it unexpectedly starts to thaw. Overarchingly I really enjoyed and learned so much from every component of the experience BTI had to offer, and appreciate how formative of program it has been as I consider my plan and ambitions for a future in scientific research.

Pyramiding for Fruit Nutritional Quality: Elucidating the genetic basis of folate accumulation utilizing tomato F2 populations

Folate (Vitamin B9), the collective name for tetrahydrofolate and its derivatives, are vital compounds required for the metabolism of nearly all organisms. Plants and many microorganisms synthesize folates de novo, however, humans lack this ability and require a dietary supplement, mostly from plant foods. Deficiencies in folate are associated with congenital abnormalities like neural tube defects, as well anemia, cardiovascular diseases, certain cancers and dementia. Folate deficiency remains a severe challenge worldwide, especially in developing countries. Due to the risks of folate deficiencies, governments and health organizations worldwide have recommended folate supplementation, leading to mandates on fortification of foods with synthetic folic acid in western countries. It is important to diversify dietary sources of folate, and biofortification has been proposed as another means of increasing intake of natural folates. The tomato fruit is a good model because of available breeding populations, such as the Solanum pennellii introgression lines (ILs). Each of the ILs contains a genetically mapped, single introgression from the green-fruited, wild species S. pennellii in the background of S. lycopersicum cv. M82. In this study, the objective was to identify quantitative trait loci associated with elevated fruit folate levels. Previous work analyzing genetic variation of folate levels in the ILs identified lines significantly higher and lower in folate compared to M82. Lines were also selected based on differences in expression of folate biosynthetic genes. The resulting data was used to generate crosses between selected ILs in order to pyramid (i.e., increase) folate levels. The F2 progeny of these crosses were screened for genetic markers bordering the introgressions and scored. A microbiological assay was then used to measure folate levels; this revealed that folate content is a complex nutritional trait. Further genotyping and gene expression analysis in these lines will be undertaken to elucidate the genetic basis of folate accumulation.

My Experience

I really enjoyed working at BTI this summer. Through the program, I was able to learn in depth about the process plant genome research. Not only did I learn widely applicable skills in the lab, but I was also able to learn about the process of applying for funding, presenting my research, and other skills crucial for a career in plant science research. I especially enjoyed the graduate school panel, where I got an in-depth view of Cornell’s graduate programs. The panelists were passionate about their research and highlighted the revolutionary research at Cornell. Activities like the seminar series were captivating and showed me the breadth of research in the plant sciences. After each seminar, I appreciated how we were able to get lunch and discuss their research with each lecturer. Working in the Giovannoni lab, every member was cordial and a pleasure to work with each day. I am especially grateful for my mentor Betsy Ampofo who gave her insightful guidance throughout the summer. Through my mentor, I was able to learn invaluable lessons in experimental design and other skills crucial in plant genome research. Leaving the BTI PGR program, I look forward to a career in scientific research.

“Assessment of the Role of the NOR and NOR-LIKE 1 Genes in Tomato Ripening”

Project Summary:

During the internship, we studied the function of NOR (NON-RIPENING) and NOR-LIKE 1 genes and their effect on tomato ripening. Tomatoes are a crucial cash crop to the world economy, however much of the fruit produced never reaches its destination because of rotting. Using manipulated versions of the NOR and NOR-LIKE 1 genes in tomato crops would increase tomato sales significantly, as more tomatoes would reach their destination without spoiling.

The NOR mutation is a naturally occurring loss of function gene mutation that no longer acts as a major ripening regulator as when mutated it blocks all ripening related processes. The NOR-LIKE1 gene shares 72% homology to the NOR gene. To explore the function of the NOR-LIKE 1 gene, an RNAi approach was previously used by others in the lab to downregulate expression.  A possible redundant function of these two very similar genes was tested by over expressing (OE) the NOR gene in the NOR-LIKE1 RNAi repression line to see if NOR could recover any of the ripening deficiencies resulting from NOR-LIKE 1 repression. Three transgenic lines for each construct were available in the green house at the start of my internship. In order to identify plants that contain transgenes DNA extractions and PCR using gene specific primers was performed. Plants that had positive PCR amplifications for the transgene were selected and transplanted for further analysis of fruit ripening phenotypes. Downregulation of the NOR-LIKE1 gene results in yellow-orange-fruit that partially ripens suggesting a role of this gene in fruit ripening.  Overexpression of the NOR gene in the NOR-LIKE1 RNAi line produced plants that present NOR-LIKE1 plant and fruit phenotypes, suggesting that the NOR gene is not able to complement the NOR-LIKE1 gene and therefore that while both related genes influence ripening, the functions of these two genes are distinct.

My Experience:

This research project taught me many important lessons, not only about research but about work and even life in general. I learned a lot about what research is like as well as molecular techniques used to perform that research. Additionally, we were taught not only lab tools and techniques but also how to make connections between experimental results and the objective of our projects. I also learned about the laboratory environment and how important is interaction with the mentor and other people in a lab in order to perform successful experiments and correct mistakes. Finally, I learned important life lessons about work. This internship was my first job, and it made me realize that hard work is necessary to accomplish anything in the work world.

“Understanding the role of SlDML2 expression on ripening initiation in tomato fruit”

Project Summary:

More than one third of fresh fruit produced in the US each year is lost due to spoilage. As the ripening process predisposes fruit to spoilage, the manipulation of ripening regulation is an attractive strategy to reduce losses and promote food security. There are three main facets of ripening regulation: transcriptional control, ethylene signaling and DNA demethylation. The least is known about DNA demethylation, although it has been shown that global DNA demethylation occurs during ripening. Also, chemically-mediated DNA hypomethylation can trigger ripening in immature fruit, suggesting that DNA methylation levels influence the timing of ripening initiation. In tomato, the gene DEMETER-LIKE DNA DEMETHYLASE 2 (SlDML2) is necessary for proper ripening, as fruit fail to ripen when its expression is inhibited. My summer project was to study the role of SlDML2 in ripening initiation using lines that overexpress SlDML2. I characterized the phenotype of these lines along with crosses made between the SlDML2 overexpression plants and several non-ripening mutants that are impaired in DNA demethylation to determine if SlDML2 overexpression could fully or partially restore ripening. While no obvious ripening phenotypes were observed, bisulfite PCR showed that methylation levels are reduced in the strongest overexpression lines indicating that, while the transgene worked, it may not have sufficiently demethylated promoter targets to be effective. I also created a CRISPR/Cas9 construct that will be used to generate an SlDML2 knockout mutant. In conclusion, our results suggest that DNA methylation levels respond to SlDML2 overexpression, although more in depth characterization of these lines is required to assess whether or not there is any ripening change in these lines. If so, this work could be used as a model strategy to control fruit ripening in additional species, thus enhancing food distribution and production systems.

My Experience:

My experience in the BTI-Cornell PGRP REU program has been incredible! I have learned so many new research skills, from plasmid isolation to creating CRISPR/Cas9 constructs, and I have gained experience with plant genetics and sequence data analysis. My mentor, Dan Evanich, was incredible in that he was very eager to ensure that I understood the details of my research, but also was willing to step back and let me carry out procedures on my own. This experience has improved my independence as a researcher and my ability to think about the broader implications of research results and has challenged me to become more familiar with various computational biology skills. My interactions with the various faculty and staff at BTI and Cornell have helped reconfirm that I want to work with plant science research and have given me insight into what I need to do to prepare for graduate school.

“Analysis of Cold Stress Responses in Solanum lycopersicoides Introgression Lines”

Project Summary:

While the world population is rapidly growing, arable acreage is shrinking causing strain on agriculture. Severe shifts in weather patterns associated with climate change further aggravate this strain. These conditions collectively threaten crop yield and food security across the globe. The demand for traits such as cold tolerance has increased because plants that express these traits would be better able to tolerate harsh conditions and meet food supply demands. These plants could also broaden the seed-to-harvest window thereby allowing farmers more flexibility, productivity and profitability.

Solanum lycopersicoides, a nightshade tomato relative, grows in dry, cold alpine regions of Peru and Chile, making it of interest for both cold and drought tolerance studies. The development of introgression lines (ILs) containing introduced chromosomal segments of S. lycopersicoides into the genetic background of the cultivated tomato S. lycopersicum could possess cold tolerance. By quantifying stress response in ILs, lines of interest can be identified for further investigation of cold tolerance mechanisms and as a direct tomato breeding resource.

I analyzed the stress responses of an IL population by analyzing their malondialdehyde (MDA), proline, chlorophyll, and brix contents. Qualitative data was additionally gathered by photographing plants as part of a survival assay.  Several ILs were determined to be potential candidates for cold tolerance where multiple assays supported this conclusion. Future work could focus on fine mapping quantitative trait loci (QTLs) that could be conferring cold tolerance.

My Experience:

My experience here at BTI has been truly invaluable. I gained hands-on experience with research techniques, analytical equipment, and experimental design. The weekly seminars, many talks with mentors/lab members, and reading primary research broadened my knowledge in various plant science topics.

My mentor and I worked closely to design and modify my summer project. He encouraged me to take the lead and conduct most parts of project myself, but he was always available if anything was unclear. This approach allowed me to analyze each protocol critically and become familiar with my project’s experimental methods. I developed a confidence in my ability to conduct meaningful research and strengthened my interest in attending graduate school. I feel ready, more than ever before, to pursue a fruitful career in plant science.

Project Summary

Ripening in fleshy fruits is a tightly regulated process aimed at making the fruit appealing to seed-dispersing organisms. The accumulation of key nutrients during ripening also makes fleshy fruits important for a balanced diet. However, unlike staple crops (e.g., maize and rice) fleshy fruits have a limited shelf life before they begin to rot. One way to address this problem is to control when the ripening process is initiated. In this study we utilized tomato, the primary model for fleshy fruit ripening. While a general model of ripening regulation in tomato has been established, the initiation of ripening is not well understood. One clue however, is that ripening begins in the locule (i.e., gel-like tissue that surrounds the seeds) and moves outward, raising the logical question of whether or not seeds initiate or influence ripening. To test this question, we employed a system for hormonal induction of parthenocarpy (i.e., fruit development in the absence of fertilization) to produce developmentally synchronized seeded/seedless fruits. We then determined if/how key ripening parameters were altered in the absence of seeds. First, we observed liquefaction of the locular gel was significantly delayed in seedless fruits, suggesting seeds may play a role in changes prior to the start of ripening. In addition, we found a significant reduction in the levels of several carotenoids in the flesh of parthenocarpic fruit, but failed to detect a difference in the production of ethylene (a key ripening hormone) during ripening. Future work will aim to better characterize the alterations in ripening observed here through transcriptomic and epigenetic approaches.

My experience:

Working at the Boyce Thompson Institute this summer has been a life changing experience. I performed many new techniques as well as learned new ways to perform techniques I had done in the past. It was also fun doing hands on work in the greenhouse while also doing molecular work in the lab.  I thoroughly enjoyed working with my mentor. He not only taught me proper lab etiquette but also showed me what it is truly like to be a graduate student. It was also a pleasure working with everyone else in the Giovannoni lab. Working forty hours a week had prepared me for graduate school as well as a career in plant science. This internship has furthered my love and admiration for plant research.

“Carotenoids quantification and quantitative trait loci (QTL) analysis in a tomato recombinant inbred line (RILs) population”

Project Summary:

Tomatoes are one of the most widely consumed fruit and a source of various nutrients in humans. However, consumers have been complaining of the decreasing flavor quality. Among the components of tomato flavor are aroma volatiles, which are produced by the catabolism of compounds such as carotenoids, namely apocarotenoids, amino acids and fatty-acids. Carotenoids have nutritional value as they serve as the pro-vitamin A, antioxidants, and can potentially decrease certain cancer types. In this project carotenoids were extracted from ripe tomato fruits of 132 recombinant inbred lines (RILs) derived from a cross between a cultivated tomato, Solanum lycopersicum (cv. NCEBR-1) and a close wild relative, S. pimpinellifolium (LA2093) and analyzed by super-critical fluid liquid-chromatography equipped with photo diode array detector. Then, carotenoids were quantified and a quantitative trait loci (QTL) analysis was performed using r/qtl to determine loci across the chromosomes accounting for carotenoids accumulation. Five out of six carotenoids showed a major QTL at the end of chromosome 12 explaining 9%-62% of the phenotypic variation, which is the location of the zeta-carotene isomerase gene, however, no QTL was co-localized with apocarotenoids QTLs at chromosome 1. Lycopene and beta-carotene, but not phytoene and zeta-carotene, shared a QTL at the same location on the beginning of chromosome 7 explaining 15% and 12% of the phenotypic variation, respectively. The causative gene for this QTL is unknown and finer mapping will be needed to determine it. Finally, this study showed that carotenoids levels are not affected by their catabolism into volatiles, but it produced some QTLs to be further investigated for both breeding and scientific purposes.

My Experience:

I enjoyed being an intern in the Giovannoni lab and getting to work alongside my mentor as well as many other accomplished scientists. I liked seeing how diverse the areas of study were even within the same lab.  I have had the opportunity to gain confidence in my ability to successfully execute lab techniques and get results. The weekly seminars exposed me to just how many areas of plant biology there are and exposed me to disciplines I was previously unaware of. Coming into this summer, I knew I wanted to do scientific research, but this experience has given me a better idea of what areas of research I would most enjoy. Additionally, the bioinformatics workshops allowed me to better understand an area of science that I previously had little experience with.

High Pigment Mutations and Environmental Responses

Project Summary

Tomatoes (Solanum lycopersicum) are widely grown crops, important for their nutritive properties. Both their economic and health applications, combined with the wide availability of genetic resources for tomato, makes it a powerful model for studying fruit development. Tomato plants possessing high-pigment mutations merge the study of fruit development with the promise of increasing fruit qualities such as nutrition and shelf life. Two mutations were considered, high-pigment 1 (hp1) and high-pigment 2 (hp2). These mutations, in different genes that confer an elevated light signal throughout the plant, result in increased pigment accumulation, delayed ripening, and dwarfed plants, among other phenotypes. Our work was to use different light and ethylene treatments on these mutants to elucidate the tangled network of signal interactions between light and ethylene. We also looked at how this network can impact fruit development, as well as nutritionally and economically important fruit traits like weight and firmness. Manipulation of light signal networks is a promising route to pursue in plant breeding.

My Experience

My experience as a Boyce Thompson Institute Intern has been extremely positive. The circle of interns is very friendly and inclusive, I have made many lasting friends. I had that same inclusive experience in the lab of Dr. Giovannoni. Because the lab is so diverse, I had the opportunity to learn and experiment with people from all around the world. It was great to further my molecular biology skills and design experiments alongside learning new techniques involved in economically important research like fruit quality. I also was able to start work on some CRISPR cloning. Learning one of the newest and most exciting molecular biology techniques as it develops was very rewarding. I feel more prepared for grad school and more confident in my ability to succeed ther after my time at BTI, and I would recommend it to any undergraduate considering work or a career in the biological sciences.

The Impact of Drought Stress on the Kinetics of Water Intake in Select

Project Summary

Drought is one of the most notorious stressors that plague our crops. Therefore, it is important to develop plants that are durable and can withstand periods of water stress. Drought response genes can be challenging to isolate, but the use of introgression lines (ILs) can mitigate this challenge somewhat by taking advantage of the fact that wild ancestors of many cultivated crops are reservoirs for stress-tolerant genes. ILs combine overlapping fragments of the wild ancestor’s genome with the genome of cultivated lines. These insertions can then be mapped and IL plants can be screened for phenotypic changes. From this information quantitative trait loci (QTLs) can be identified and the functions of the genes in the inserted regions can be deduced.

In order to try to isolate drought resistant QTLs we have screened selected lines from thelycopersicoides IL population. These lines were subjected to drought stress conditions and then were recovered from stress. During this process, the kinetics of water intake were monitored and differences between some of the ILs were observed. The outcome of this research and subsequent research should help provide a better understanding of the basic mechanisms governing drought tolerance in tomato. It will also supply useful tools for plant breeders as they tailor new varieties of crops to cope with the climate change and population demands of the twenty-first century.

My Experience

My ten weeks at Boyce Thompson Institute (BTI) have been invaluable. I have gained many insights into the research process and have met and gotten to work alongside so many inspiring and dedicated researchers. I thoroughly enjoyed the time I have spent working in the Giovannoni lab and with my mentor, Ari Feder. Ari has been a fantastic guide this summer—providing guidance and support but also letting me be independent at times. I have learned a number of valuable lab techniques, analyzed data, solved problems, but most importantly, I have grown more confident as a biologist. It has been delightful to get to be a part of the important work that is conducted at BTI and I have developed a new perspective on the importance of research in the plant sciences. Although my time at BTI has come to a close, I will be able to take the skills I learned with me throughout my career as a scientist.

Carotenoids profiling and quantitative trait loci (QTLs) detection in a S. lycopersicum × S. galapagense recombinant inbred lines populations

Project Summary

Many people suffer from malnutrition due to lack of access to quality food, especially in developing countries. Carotenoids accounting for the red, orange, and yellow colors of tomato fruits, serve as important nutritional components such as pro-vitamin A (mainly β-carotene, orange color) and antioxidants (mainly lycopene, red color). Being able to map quantitative trait loci (QTL) for desired carotenoids holds the potential to grow more nutritious tomatoes and other fruits. We used a recombinant inbred line population derived from a cross between S. lycopersicum (a cultivated red fruit tomato) and S. galapagense (a wild orange fruit tomato). Ripe fruits were harvested and used for carotenoid extraction and RNAseq analysis. Carotenoids were extracted using methanol, tetrahydrofuran and petroleum ether and resolved by high performance super-critical fluid chromatography. As expected, the main carotenoids were β-carotene in S. galapagense and lycopene in S. lycopersicum. Some lines accumulated carotenoids in similar levels to one of the parents, but others accumulated higher levels of upstream or downstream compounds, such as phytoene or lutein, respectively. QTL analysis revealed a large-effect QTL for both lycopene and β-carotene on chromosome 6 located six genes from the lycopene beta-cyclase (SILYCB) gene. SILYCB is the enzyme converting lycopene to β-carotene. Minor QTL for β-carotene was located on chromosome 2. QTLs were found also for phytoene, lutein, and some unidentified carotenoids. This work demonstrates the power of high resolution QTL analysis and while the major QTL is of known function it can bear the potential for mining genes that underline minor QTLs.

My Experience

In the lab I work in back in Clemson, I did a lot of genetics work. This summer, I learned new techniques about how a biochemistry lab is run, that I am excited to bring back to my lab when we start getting to more biochemistry processes. Personally, I’ve gained a lot of new skills and life experiences that I would not have learned anywhere else. It gave me an insight into what I want to do with my life and the process I need to take to get there. Professionally, I gained confidence in speaking to others about my research, especially my science peers and professors. Academically, I learned I do want to go back to school and get my Ph.D. in plant breeding, however I want to spend a few years working under someone and getting the basics down. I want to thank the PGRP internship for this insightful and exciting summer

The Relationship Between Water Stress and Fruit Softening: The Cuticle as a Key Factor

Project Summary

The plant cuticle plays a critical role in preventing desiccation, but many aspects of its formation and structure-function relationships are poorly understood. Fruit of the tomato mutant delayed fruit deterioration (dfd) have exceptionally long shelf life and resist desiccation for many months after ripening. We are currently investigating whether different water regimes affect the composition and properties of fruit cuticles from dfd and from the cultivar Ailsa Craig (AC), which has normally softening fruit. It is anticipated that a better understanding of cuticle development in the context of responses to water stress will provide new insights into the molecular bases of prolonged shelf life in fleshy fruits.

My Experience

I decided to apply for an internship at BTI because I wanted to see what it was like to perform research at a top institution, and what it was like to work in a molecular lab since my background is in the ecology and evolution of plants. The internship has fulfilled both of these goals in strides, and has helped me formulate ideas on what the next step in my academic career will be. Even though I am going to pursue my original interests in the ecology of plants, the molecular lab skills I gained from the summer internship will allow me to work on a larger range of research questions.

Tracing Gene Expression amongst Constituent Cell Types in the Pericarp of Tomato (Solanum lycopersicum)

Project Summary

The tomato (Solanum lycopersicum), is a universally preeminent basis for fruit study and research and is an important component of the human diet. Previous research studying gene expression in the tomato fruit has almost entirely focused on the pericarp as a collective whole as opposed to differentiating the tissue into the individual constituent cell types and tracing the activity of each separately. Using the undivided pericarp provides an average of gene expression levels, at best, whereas isolating the components should create a more specific understanding of fruit development, diversity, and response to stress as well as provide a clearer understanding of the distinct levels of gene expression in specific tissues and at various stages of fruit development. This can potentially lead to innovations in shelf-life, yield and nutritional quality with benefit for humankind in economics, health, and sustainable production. My project primarily focuses on genetically profiling the genes located in the distinct tissues of the pericarp of the tomato fruit throughout the different stages of development using three principal steps: RNA extraction, strand-specific RNA sequencing library construction, and RNA sequencing, which will enhance previous NSF studies in tomato genomics and create a template for further research in model agricultural and commercially important plant species. A substantially more complete picture of fruit development should also result from the data collected from this project, as well as a new platform for biological discovery.

My Experience

This summer’s experience at the BTI has taught me valuable lessons in both lab techniques and in working in a professional setting. I would like to thank everyone in the PGRP internship program and the Giovannoni Lab for being such a welcoming group and making my experience such an enjoyable one. My mentor, Yimin Xu, has prompted me to have greater confidence in myself by encouraging the belief that mistakes are opportunities for learning and teaching. The workplace environment was such a friendly one that I never hesitated to ask questions and there was never a day I didn’t look forward to coming to work. I was given advantageous experience in key research techniques that will benefit me as I pursue studies as an undergrad and graduate student. BTI’s summer internship program has been such a rewarding experience that I wouldn’t hesitate in joining again and suggesting enthusiastically to others.

Quantification of Folate Content in Solanum lycopersicum × Solanum pimpinellifolium Recombinant Inbred Lines (RILs) for QTL Analysis Using Microbiological Assay

Project Summary

Deficiency of folate and its derivatives (vitamin B₉) is a worldwide problem that can impact human health, causing birth defects such as spina bifida, as well as a higher risk of cancer, cardiovascular disease and anemia. Fruits constitute important components of the human diet and contribute a large portion of vitamins, minerals, antioxidants and fiber. All of these fuel the interest for the enhancement of folate content in crops. According to the United States Department of Agriculture, tomato is the second most consumed vegetable in the U.S., but provides roughly 4% of the recommended dietary allowance of folate. The focus of this project is to identify candidate genes affecting folate production using mapping data of a recombinant inbred lines (RILs) population to generate quantitative trait loci (QTLs). Total folate content was determined in 85 lines of a Solanum lycopersicum (NCEBR1) × Solanum pimpinellifolium (LA2093) RIL population using a microbiological assay, utilizing Lactobacillus rhamnosus which requires folate for its growth. We found that the parents of the population show differences in their folate content. S. lycopersicum contains 9.2 µg/100 gFW, while the wild S. pimpinellifolium contains 26.4 µg/100 gFW. Furthermore, in a preliminary LCMS analysis of selected RILs there is a correlation between high folate content and glutamate levels, and between low folate content to glutamine levels. Based on these data, QTLs controlling folate levels will be generated. These QTLs, in conjunction with RNA-seq data, will be used to facilitate the identification of candidate genes governing folate content in tomatoes fruits.

My Experience

Participating in this program has been an extraordinary experience. I was placed in the Giovannoni lab where I worked alongside amazing mentors who ensured I got the best out of my participation there. The lab environment was very pleasant and kept me motivated throughout the 10 weeks. My mentors provided the right guidance I needed for the project but also allowed me to work independently which boosted my confidence in my ability to conduct research. This internship helped me both fortify my interest in a career in plant sciences as well as further affirm its importance and worldwide contribution, and to finally feel prepared for graduate studies.

Fine tuning of the Solanum lycopersicoides introgression population and carotenoid analysis of selected lines.

The tomato fruit is one of the most consumed vegetables in the world, however its nutritional values has decreased in the past decades as an undesirable result of selection for greater yield and longer shelf life. In order to enhance nutritional properties in tomato, current efforts are focusing on increasing the levels of amino acids, vitamins, micronutrients and antioxidants. Tomato introgression lines were proved to serve as a powerful tool in exploiting natural genetic variation, enabling introduction of desired traits into cultivated lines. S. lycopersicoides potentially possess such an important genetic pool.  Not much work has been conducted on this population due to high heterozygosity and multiple insertions. In order to cope with this complexity, we have developed genetic markers to screen and fine tune the population. In addition, carotenoid analysis has been conducted on fruits of selected lines within the population to identify associated QTLs. This work serves as the basis for further extensive genetic and biochemical screening of the population which will enable to exploit its potential.

My Experience

Working with Dr. Giovannoni’s lab team was a unique experience for many different reasons. First, it is a multicultural lab and I was able to meet people from all around the world, with different cultural backgrounds and ways of not only thinking, but also problem solving. This, in my opinion, helped me to improve myself as a future researcher, team member, and even as a better person. In addition, Dr. Giovannoni has a brilliant way to lead the lab group, encouraging independence in research and at the same time providing a pleasant working environment. Throughout my time in the lab I had a chance to be engaged in the preparation and management of a tomato field, all the while learning current approaches in molecular biology and biotechnology as a tool to understand the novels in tomato ripening. In the end, BTI provided everything that I needed, and everybody in the department was always willing to help. Working at BTI increased my passion for research.

Investigating FBOX protein interactions with ethylene receptors via targeted yeast two hybrid assays

Project Summary

The final stages of fruit development culminate with ripening and senescence and coincide with a burst of respiration and ethylene in fruit classified as climacteric. Ethylene is a gaseous plant hormone that regulates ripening and other aspects of plant development such as senescence, abscission, biotic and abiotic stress responses, and seedling growth, thus its action is tightly controlled by a complex biosynthesis, perception, and signal transduction system. While the synthesis, perception, and signal transduction is well characterized in plants at the transcription level, little is known about post-translational regulation of these pathways. There is evidence in the literature that the ethylene receptor levels are controlled by protein degradation via the 26S proteasome pathway.  Two genes encoding FBOX proteins have been identified as candidates involved in this regulation in tomato, (SlFBOX1 and SlFBOX2), and the expression of these genes is up-regulated in ripening fruit. Plants silenced for SlFBOX1 and SlFBOX2 exhibit altered ethylene responses and ethylene synthesis as well as accumulation of the ethylene receptor proteins ETR3 and ETR4. FBOX proteins are involved in protein turnover by recruiting target proteins for degradation via the 26S proteasome. We hypothesize that SlFBOX1 and SlFBOX2 are involved in the turnover of ETR3 and ETR4 protein levels. This hypothesis will be tested by using a targeted yeast two hybrid assay to assess whether or not SlFBOX1 and/or SlFBOX2 interact directly with ETR3 and/or ETR4. The results of these targeted yeast two hybrid experiments will be reported and discussed.

My Experience

My Experience at BTI as a PGRP intern has given me the opportunity to delve deeper into plant research and gain essential laboratory experience. Specifically, I had the opportunity to perform yeast two hybrid assays and investigate protein-protein interactions in tomato. A warm welcome by the Giovannoni lab made it easy for me to jump right in and begin work on my summer project. The most important things I learned at BTI were how valuable patience and learning from previous mistakes are in scientific research. This internship  solidified my interest in going to graduate school to study plant breeding and to have a career in plant research.

Development of nearly isogenic lines of tomato for the gene conferring the green shoulder phenotype

The tomato uniform ripening (u) mutation confers a light green fruit phenotype as compared the wild type (U) green shoulder and is widely used in breeding for tomato varieties producing evenly ripened fruit. U encodes a GLK transcription factor influencing chlorophyll accumulation and distribution in developing fruit. Tomato GLK influences fruit chloroplast development and fruit quality and manipulating GLK expression is a means to enhance fruit quality and nutritional value. Most commercial tomato varieties have the u mutation and hence non-functional GLK protein. One approach to manipulate GLK expression is to bring back the normal green shoulder phenotype to elite cultivars using traditional breeding techniques. The introgression line 10-1 of Solanum pennellii in the cultivated tomato M82 has the green shoulder phenotype but also contains a large segment of chromosome 10 from the wild species that may include genes that could modify characters other than the green shoulder phenotype targeted here. I screened a large segregating population derived from this introgression line to identify recombination events near the U locus and successfully identified several plants with smaller introgressed fragment sizes. Homozygous progeny from these plants will be compared with the M82 line for sugar and carotenoids to test the effect of the green shoulder phenotype absent changes that might result from additional linked wild species alleles.

My Experience

My overall experience here in the Giovannoni lab was great. The environment and lab setting has giving me the confidence to continue research. Along with learning new techniques I feel that I will benefit from and can bring along with me in any research I may conduct in the future. Although I’ve learned plenty of different things in the lab, I feel that one thing that I’ve learned is more patience. Working one-on-one with my mentor and other faculty members has giving me the opportunity to troubleshoot experiments. My mentor has also giving me the chance to work independently and stressed the importance of being precise in scientific tests. This being my first internship, I’ve learned a new level of respect and the hard work that goes into the production of conclusive data in science.

Carotenoids are 40- carbon lipid-soluble terpenoid molecules which are present in most tissues of higher plants. Carotenoids are strong antioxidants that protect the plant from harmful reactive oxygen species (ROS) and protect plastids from photo-oxidation from excess light energy. Carotenoids also play a role in human health. Studies show that carotenoids such as lutein, lycopene, and b-carotene can lower the risk of certain types of cancer and prevent blindness and diseases of the eye. Carotenoids are also a precursor to the hormone abscisic acid (ABA) in plants, which is important in protecting the plant from abiotic stresses (i.e. drought, water stress, etc.). Galpaz et. al (2008) showed that the loss of ABA in developing fruit leads to increased chloroplast division. In this study we wanted to explore this relationship further by comparing the ABA deficient mutant, notabilis (not/not) and two independent transgenic ZDS-RNAi lines, which present a block at zeta-carotene desaturase (ZDS) in the carotenoid biosynthesis pathway and low ABA levels, to wild type (Solanum lycopersicum, cv. Ailsa craig (AC)). ZDS is located upstream of the photoprotecting carotenoids and ABA biosynthesis, whilenot does not affect carotenoid biosynthesis directly and is specific to ABA biosynthesis, therefore physiological changes in these lines are very different from each other and from AC. Our goal was to compare the type and amount of carotenoids, chlorophyll, and hormones produced in each of the three genotypes throughout the stages of fruit development and to see how changes in these biochemical profiles affect the fruit as it proceeds to ripening.

My Experience

My 10 week experience with the Giovannoni Lab at BTI was very beneficial in a few different ways. After my time here, I feel much more confident in my abilities to work in a lab, conduct research, and apply to graduate schools that I never thought I would have had a chance of getting into before. My mentor was very helpful when I needed guidance, but was also gracious enough to let me work alone when I was comfortable with a protocol so as to help build my confidence in my abilities. I was also able to truly see what it is like to work in a lab environment on a 9-5 basis, and have a much more positive attitude towards doing this in the future. I came into this internship as a full-fledged environmental science major, not very comfortable with, or excited about, molecular biology. However, I honestly have to say that after this summer, I am considering a future career in something more lab-oriented and molecular-based.

Complementation of Vitamin B6 Mutations in Arabidopsis thaliana and Saccharomyces cerevisiae with a Ripening Related SlPDX1 cDNA from Tomato

Ethylene is an important plant hormone that regulates many physiological processes in plants, including ripening in climacteric fruit. Understanding the molecular events that regulate the biosynthesis and signal transduction of this hormone then is of great importance, not only for tomato (Solanum lycopersicum), the model system for fruit ripening, but for many other species of economic importance. ACC synthase is the rate limiting step of ethylene biosynthesis and its activity requires vitamin B6 as a co-factor. SlPDX1 encodes a putative enzyme involved with the biosynthesis of this co-factor in tomato. Tomato plants were previously generated using RNAi to silence SlPDX1. The resulting plants exhibit a delay and inhibition of ripening and reduced ethylene. In addition, both vitamin B6 and ethylene treatment rescues the inhibition of ripening phenotype, resulting in normally ripened fruit. Our main goal was to determine if the SlPDX1 sequence encodes an active enzyme by complemention of vitamin B6 biosynthetic mutations in either A. thaliana or S. cerevisiae. We amplified SlPDX1 from tomato fruit RNA using RT-PCR, and the resulting amplicons were sequenced then ligated into appropriate plasmid vectors for expression in both A. thaliana and S. cerevisiae. The subsequent constructs were deployed for the appropriate species, the results of which will be reported and discussed.

My Experience

During my time here, I’ve learned techniques that I otherwise might not have been exposed to. I’ve grown an appreciation for Boyce Thompson Institute’s mission and plant research as a whole. It was incredibly rewarding when hard work and patience got me the results I needed but I learned that getting results is a process. Working full-time on a project has taught me how to deal with roadblocks when they arise by reflecting on past work to circumvent the problem. This program has reinforced my interest in scientific research.

Accurate Profiling of the Tomato Fruit Transcriptome

Project Summary

This past summer, I used Next Generation RNA Sequencing technology to profile the tomato fruit transcriptome. An understanding of the gene expression pattern throughout fruit development will help elucidate which transcripts are involved in the ripening process. We focused on improving the existing RNA-seq methodologies to develop a novel, bias- free, and strand specific RNA-seq protocol (AF ssRNA-seq). AF ssRNA-seq enables me to accurately achieve the transcript accumulation at a certain stage during tomato fruit development.

My Experience

This PGRP internship experience was very informative and enjoyable. I found it easy to adapt to the environment in the BTI facility and in the lab. The Giovannoni lab was welcoming; each member was approachable and kind in answering any question I had. The PGRP internship program gives each intern the opportunity to work one on one with a faculty mentor. My mentor was very helpful and provided detailed explanations in regards to every experiment I performed. My overall understanding of molecular biology has expanded greatly. This internship was great preparation for grad school because I was far from my comfort zone in a new laboratory setting and managed to learn a great deal.

Designing Functional Ripening-Control Tomato Promoters

Tomato, Solanum lycopersicum has been the subject of many studies focused on the development and ripening process of climacteric fruits; studies focus on pigmentation, texture, aroma, flavor, and nutrient composition (Moore et al., 2002). To study fruit ripening, it is necessary to use ripening mutants. Two naturally occurring recessive mutants ripening inhibitor (RIN) belongs to the MADS-box gene family and another mutant non-ripening (NOR) belongs to the NAC family. Both gene are transcription factors that control ripening.

The goal of this internship was to design functional RIN and NOR promoter constructs. Four promoter constructs were designed for NOR and three were designed for RIN to elucidate the most efficient constructs. Promoter constructs are assembled within the pBi101 transformation vector using GUS gene expression assay. Constructs were created with at least 3.2 Kb of promoter sequence upstream of the 5′ translational start site. Promoter regions were paired with ‘required’ intron regions of DNA to include possible cis-regulatory elements within constructs to achieve high expression level. These functional promoters will be used as a tool for genes that need to be expressed in a fruit-specific manner.

My Experience

Overall, the internship was a great success. I was lucky enough to be in a lab with two other interns. The lab environment was surprisingly laid-back and very easy to integrate into. All the post-docs, research associates, and graduates students were excellent resources for my project and for my growing curiosity in graduate degree programs. Working directly with a mentor on my research was beneficial in learning new scientific techniques and for gaining an understanding of the dedication a scientific career requires. Through my experience and interaction with my peers I was able to gain an accurate view of the career paths available in plant research and glimpse the projects graduate students focus on during the PhD research.

Characterizing the sherry mutant in Solanum lycopersicum (cv. Canary Export)

For my summer internship project, I worked on characterizing the sherry mutant in solanum lycopersicum cv. Canary Export. For this project we wanted to learn more about the sherry mutant, which was created through fast neutron mutagenesis. The sherry mutant was selected from a carotenoid mutant accumulation assessment screening, and was selected as a good candidate for further research to better elucidate the fruit ripening process in the tomato. What I worked on specifically was looking at sherry’s ethylene sensitivity response via the triple response assay, as well as looking at sherry’s carotenoid profile using HPLC separation, to see how it differed from it’s wildtype background. I then took that profile and looked at the gene expression through Real Time PCR, in the carotenoid biosynthetic pathway of our sherry mutant, to see if maybe that played a role in the mutant phenotype that we were seeing. We also wanted to look at other aspects of ripening, to determine if sherry was somehow controlling more then just carotenoid accumulation, and we did this by looking at the levels of ascorbic acid accumulation through ripening in our sherry mutant vs. Canary Export. We came to the conclusion that the sherry mutant was indeed effecting other aspects of fruit ripening, other then just carotenoid accumulation. Other work that needs to be done for this project: the sherry mutant needs to be mapped to find out exactly where the mutation is located, it is currently punitively mapped to chromosome 10, as well as backcross sherry with the Alisa Craig background create a near isogenic line (NIL) and use this to compare it to other ethylene mutants. Overall this has definitely been one of my most exciting learning experiences of my life, and I am so very grateful for the opportunity to have worked along side prestigious scientists such as Jim Giovannoni. The knowledge I learned this summer is invaluable to my future career as a researcher, and I thank the PGRP program for this awesome learning opportunity!

My Experience

For my summer internship project, I worked on characterizing the sherry mutant in solanum lycopersicum cv. Canary Export. For this project we wanted to learn more about the sherry mutant, which was created through fast neutron mutagenesis. The sherry mutant was selected from a carotenoid mutant accumulation assessment screening, and was selected as a good candidate for further research to better elucidate the fruit ripening process in the tomato. Overall this has definitely been one of my most exciting learning experiences of my life, and I am so very grateful for the opportunity to have worked along side prestigious scientists such as Jim Giovannoni. The knowledge I learned this summer is invaluable to my future career as a researcher, and I thank the PGRP program for this awesome learning opportunity!

Mapping and Characterization of nei mutant in Solanum lycopersicum

The tomato, Solanum lycopersicum, is one of the United States largest economic vegetable crops and is currently the most commonly used plant model system for studying the process of fruit ripening. One way to study a gene’s function in fruit ripening is to observe its lack of function when the gene contains a deleterious mutation.

Identification and characterization of a quantitative trait locus of B-carotene in the Solanum penellii introgression line IL2-2

Synthesized in the plastid, carotenoids are crucial actors in photosynthesis as antioxidants, fighting photo-oxidative damage. Tomatoes primarily contain the red-pigmented lycopene, which is a pre-cursor to b-carotene (provitamin A) and a-carotene. This summer, I sought to both identify and characterize a b-carotene regulating QTL in the S. pennellii line IL2-2.

Characterization of ethylene signal transduction genes through RNAi gene silencing

Understanding the complexity of ethylene signal transduction and regulation in regards to fruit ripening is extremely important, particularly from the economic standpoint of genetic manipulations for produce improvement. With RNAi technology it is now possible to reduce the level of expression of a targeted gene through post-transcriptional gene silencing in plants. By using this technology to reduce expression of the genes in the ethylene signal transduction pathway, individual functions of these genes can be characterized.

Microarray analysis of differential gene expression in tomato apricot mutants

The tomato mutant apricot is a putative transcription factor controlling the ripening of tomatoes, especially the carotenoid, folate, and ascorbate biosynthetic pathways. My presentation will focus on the microarray experiments carried out this summer assaying for differential gene expression between wild type Ailsa craig tomatoes and apricot mutants. qRT-PCR was also carried out, confirming the results obtained from the microarray experiments.

Jessica is a student at California State University . In the Giovannoni lab, Jessica’s summer internship focused on identify gene promoters that will switch on during mid to late stages of the ripening process using standard molecular techniques such as DNA extraction, gene sequencing, bioinformatics, bacterial DNA transformations, and inverse PCR under the guidence of post-doctoral associate Nigel Gapper.

Catherine is a student at Williams College. In the Giovannoni lab, Catherine’s summer internship focused on characterization of the Tomato NAC Gene Family using standard molecular techniques such as bacterial DNA preparation, gene sequencing, bioinformatics, real-time Polymerase Chain Reaction (PCR), and phylogenic analysis under the guidence of post-doctoral associate Julia Vrebalov.

During the summer Maria was a recent graduate at Cal State University- Fresno in California. She worked in the Giovannoni lab with graduate student, Catherine Martel on development of DNA constructs for yeast one-hybrid screens for the NOR fruit ripening promoter. During her summer at the BTI, Maria learned bacterial DNA preparation, creation of DNA constructs, PCR and yeast transformation.

Stephanie is currently a student at Ithaca High School. This summer, Stephanie worked with graduate student Elizabeth Fox analyzing the genetic differences between a wild type tomato plant and certain mutants. A better understanding of the genetic make-ups of the tomato and the various gene functions may eventually lead to improved yields and nutritional value of this popular fruit.

Although Stephanie enjoys playing violin, piano, and tiddlywinks, and swims a mile each day, her greatest interest has always been in the biological sciences. Stephanie reports, “This great opportunity to participate first hand in professional research has provided me with greater insight on my future career plans. I am very grateful to the scientists in my lab who have been very nurturing to me. I enjoyed working with them immensely.”