Anna Stepanova

Overview. Changes in gene expression are at the core of many biological processes, from forming a multicellular organism from a fertilized egg to surviving pathogen attacks or coping with environmental pressures. Although transcription regulation plays a critical role in modulating gene expression, growing lines of evidence indicate that gene-specific regulation at the translational level is also critical for many of these important biological processes. Unfortunately, the existing technologies to quantify changes in translation, both at genome-wide and single-gene levels, are technically demanding and costly, thus hindering the widespread investigation of this type of regulation. The development of technologies that make the quantification of translation efficiency routine has the potential to transform the field of gene regulation, allowing for the discovery of many more processes and genes regulated at the translational level. This, in turn, will open new opportunities to manipulate gene expression for both basic and applied purposes. Currently, the most widely used approach to determine the translation level of a gene is the expensive and technically demanding ribosome profiling (aka Ribo-seq) which involves quantifying the levels of each transcript and the corresponding number of associated ribosomes. We argue that this information could also be obtained by a much simpler process of determining the position of just the first or last ribosome (most 5’ or most 3’) in each transcript and then comparing the distribution of these first or last positions between two different experimental conditions. Although in principle, there is no conceptual reason to think that this Ribosome Position Inference (RiboPI) approach would not work, critical technical unknowns make this a high-risk, high-reward proposal. Intellectual merit. The main objective of this proposal is to develop an efficient, simple, and scalable RiboPI technology to quantify translation rates at both genome-wide and single-gene levels. If successful, RiboPI will make translation regulation information as accessible as RNA-seq did for transcriptomics, reducing the cost and time requirements, the complexity of the experimental procedures, and the amount of biological material needed. Not only will this make translation analysis a routine technique in many labs, but it could also bypass some of the limitations of the current technologies, such as the difficulty of mapping the very short ribosome footprints to specific splice variants, alleles, or even homeologs in polyploid species, or enable targeted studies for a group of genes. To achieve this goal, we propose to develop RiboPI, an experimentally simple approach to capture the position of the first or last ribosome in each transcript and computational methods to compare the distribution of these ribosome positions between different experimental conditions to estimate translational levels from this information. The proposed experimental pipeline involves testing novel combinations of in vivo and in vitro molecular biology procedures to efficiently and specifically map the first/last ribosome in a transcript. Some of the unknowns that make this proposal high-risk are (1) the uncertainty of whether suitable experimental conditions can be found (e.g., that preserve ribosome binding and promote reverse transcriptase activity but melt the secondary structure of mRNA) and (2) the ability to infer the efficiency of translation from the distributions of first/last ribosomes on transcripts. By comparing results obtained with classical Ribo-seq to those obtained with RiboPI, we will be able to determine the reliability of the new approach. Broader impacts. In addition to the clear benefits of developing a new experimental approach to quantify gene-specific translation efficiency and popularizing this type of analysis, this project will provide an ideal training platform for undergraduate students to experience first-hand the translation of basic biological knowledge into potentially transformative new technologies.

Phytohormones are key regulators of plant growth and development that control nearly every aspect of plant’s life, from embryo development to fruit ripening, from organogenesis to pathogen response. By altering the levels and distribution of hormones, plants can change their growth patterns and adapt to different environments, a phenomenon known as phenotypic plasticity. An overarching goal of my research is to understand how plants employ a limited set of hormones to integrate developmental programs with a wide array of environmental signals and produce adequate responses that enable the plants to survive and reproduce in even hostile conditions. I have been using various molecular, genetic, genomic, biochemical, and cell biology approaches in Arabidopsis and other plant species to explore the role of plant hormones in mediating plant phenotypic plasticity, to decipher the molecular mechanisms of auxin biosynthesis and ethylene signaling, to uncover the interaction nodes between the hormonal pathways, and to determine the contribution of translational regulation in hormone signaling and response. Despite the availability of a wide variety of biotechnological tools to manipulate plant growth, it has been challenging to precisely control when and where hormones are produced in a plant. We are developing a new set of CRISPR-based synthetic genetic devices to target expression of genes of interest to specific cell types. The potential utility of this new approach extends far beyond plants.

Title: Transcriptional and translational regulatory networks of hormone signal integration in tomato and Arabidopsis. PI: Jose M. Alonso (Plant Biology, NCSU), Co-PIs:Anna Stepanova (Plant Biology, NCSU), Steffen Heber (Computer Science, NCSU), Cranos Williams (Electric Engineering, NCSU). Overview: Plants, as sessile organisms, need to constantly adjust their intrinsic growth and developmental programs to the environmental conditions. These environmentally triggered “adjustments“ often involve changes in the developmentally predefined patterns of one or more hormone activities. In turn, these hormonal changes result in alterations at the gene expression level and the concurrent alterations of the cellular activities. In general, these hormone-mediated regulatory functions are achieved, at least in part, by modulating the transcriptional activity of hundreds of genes. The study of these transcriptional regulatory networks not only provides a conceptual framework to understand the fundamental biology behind these hormone-mediated processes, but also the molecular tools needed to accelerate the progress of modern agriculture. Although often overlooked, understanding of the translational regulatory networks behind complex biological processes has the potential to empower similar advances in both basic and applied plant biology arenas. By taking advantage of the recently developed ribosome footprinting technology, genome-wide changes in translation activity in response to ethylene were quantified at codon resolution, and new translational regulatory elements have been identified in Arabidopsis. Importantly, the detailed characterization of one of the regulatory elements identified indicates that this regulation is NOT miRNA dependent, and that the identified regulatory element is also responsive to the plant hormone auxin, suggesting a role in the interaction between these two plant hormones. These findings not only confirm the basic biological importance of translational regulation and its potential as a signal integration mechanism, but also open new avenues to identifying, characterizing and utilizing additional regulatory modules in plants species of economic importance. Towards that general goal, a plant-optimized ribosome footprinting methodology will be deployed to examine the translation landscape of two plant species, tomato and Arabidopsis, in response to two plant hormones, ethylene and auxin. A time-course experiment will be performed to maximize the detection sensitivity (strong vs. weak) and diversity (early vs. late activation) of additional translational regulatory elements. The large amount and dynamic nature of the generated data will be also utilized to generate hierarchical transcriptional and translational interaction networks between these two hormones and to explore the possible use of these types of diverse information to identify key regulatory nodes. Finally, the comparison between two plant species will provide critical information on the conservation of the regulatory elements identified and, thus, inform research on future practical applications. Intellectual merit: The identification and characterization of signal integration hubs and cis-regulatory elements of translation will allow not only to better understand how information from different origins (environment and developmental programs) are integrated, but also to devise new strategies to control this flow for the advance of agriculture. Broader Impacts: A new outreach program to promote interest among middle and high school kids in combining biology, computers, and engineering. We will use our current NSF-supported Plants4kids platform (ref) with a web-based bilingual divulgation tools, monthly demos at the science museum and local schools to implement this new outreach program. Examples of demonstration modules will include comparison between simple electronic and genetic circuits.

In the last two decades, biological research has had an emphasis on deciphering the sequence of whole genomes and on starting to identify the genetic variants responsible for the phenotypic diversity in plant and animal species. This information, together with the development and constant improvement of genome editing techniques, is making a profound impact not only on the way researchers approach fundamental biological questions, but also on how this basic knowledge can be translated into agricultural and medical practical applications. At the foundation of the current genome editing approaches is the ability of a cell to precisely replace/repair a given sequence in the genome with a repair template sequence that shares some, but often not all, of the same sequence by means of a process called homologous recombination (HR). In some organisms, such as S. cerevisiae, the high innate rates of HR can be harnessed by researchers to introduce precise changes in the genome sequence. In most organisms, however, the natural rates of HR are too low to be of practical use in genome editing. To bypass these limitations, several methods to enhance the rates of HR have been developed expanding genome editing to a wide range of organisms. One such way to enhance the rates of HR is by means of introducing double-stranded (ds) DNA breaks in the proximity of the sequence to be modified in the genome. With the recent development of easy-to-program nucleases such as Zinc finger and TALE nucleases and the CRISPR-Cas based systems, these types of approaches have gained popularity among researchers. There are, however, other strategies to enhance HR that do not rely on introducing dsDNA breaks in the genome. Among these approaches, one of the most widely used methods is the Lambda-Red system based on the expression of a set of proteins from the bacteriophage Lambda. Although this system has proven to be extremely efficient, so far, its application has been restricted to bacterial systems.

Conference Proposal: The 31st International Conference on Arabidopsis Research (ICAR2020), July 6-10, 2020, Seattle WA Rationale: Historically, ICAR has been the annual plant biology meeting attended almost exclusively by basic plant scientists working on Arabidopsis. Given the recent shift in funding towards more applied areas of plant sciences and a growing interest among Arabidopsis researchers in translational work, the focus of ICAR has also evolved to incorporate new topics and extend the impact of the conference from presenting traditional foundational studies to also highlighting new technology development, imaging, computational modeling, and practical applications. The overarching theme of ICAR2020 is Arabidopsis as a nexus for innovation, application, and impact. A broader representation of non-Arabidopsis models and crop plants at ICAR2020 and the involvement of the global plant biology community are reflected in a highly diverse list of confirmed invited speakers and the breadth of community-organized sessions. Overall goal: The goal of ICAR2020 is to bring together plant scientists working on a wide array of basic and applied questions, to encourage forward-looking conversations and intellectual exchange among conference participants, to refuel existing collaborations, to jump-start new team efforts, and to offer professional development opportunities and hands-on training in emerging areas of science via workshops. Specific objectives: The key objective of the conference is to provide a suitable platform for plant biologists of all career stages and training levels and specializing in different areas of plant science to share their latest breakthroughs, to jointly brainstorm forward-looking ideas, to devise new ways to translate the latest discoveries to practical applications and move state-of-the-art technologies to agriculturally important plant species, as well as to train and empower early-career investigators through a series of professional development workshops. Approach: ICAR2020 will feature two keynote presentations, 21 plenary talks in 7 thematic areas, 24 concurrent mini-symposia, 7 workshops, three poster presentation sessions, and several social events. While the keynote and plenary speakers were selected by the North American Arabidopsis Steering Committee (NAASC) and its External Advisory Board (EAB) while taking into account community input via an online survey, all symposia and workshop topics and speakers were suggested by the broad plant biology community through a competitive submission process. Of the 88 community-submitted proposals, 32 projects were selected and their proposers invited to organize mini-symposia or workshops. Several of the platform talks in the ICAR2020 program are directly relevant to the USDA program area priorities A1152 (Physiology of Agricultural Plants) and A1103 (Foundational Knowledge of Plant Products). Specifically, the work of ten invited speakers is well aligned with these USDA focus areas: Pamela Ronald’s research on plant immunity in rice; Tim Kelliher’s work on haploid induction in maize and wheat; Lisa Ainsworth’s studies on plant architecture, yield, and abiotic stress tolerance in a variety of crops; Ksenia Krasileva’s project on wheat responses to fungal pathogens; Polly Hsu’s work on translational regulation of stress responses in tomato; Elizabeth Sattely’s metabolic engineering efforts in tomato, cabbage, mayapple, and Arabidopsis; Robyn Roberts’ studies of the bacterial speck disease in tomato; Makenzie Mabry’s inquiry into polyploidy in Brassicas, Sam Leiboff’s research on drought responses of maize tassels and sorghum panicles; and Andrew Gloss’s work on the co-evolution of Brassicas and herbivorous flies. Potential impact and expected outcomes: USDA support is requested to partially cover travel expenses ($500-1000 per person) for the ten aforementioned plenary and mini-symposium speakers and six early-career researchers who will be selected by the organizers through a competitive application proces