Anna Stepanova

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.

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.

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

The main goal of this project is to generate a series of optimized synthetic transcriptional reporters to simultaneously monitor the activity of up to nine major plant hormones (auxin, ethylene, ABA, cytokinins, gibberellins, brassinosteroids, salicylic acid, jasmonate, and strigolactones) in a single plant. Single-hormone synthetic reporters (e.g., DR5 and EBS) have been shown to work across a broad range of plant species, making the proposed new tool useful for many plant species, from Arabidopsis to tomato and maize. By applying the synthetic biology principles of standardization and reusability to all ????????????????genetic parts??????????????? (e.g., synthetic minimal promoters, CDSs, or terminators) and ????????????????modules??????????????? (whole transcriptional units, or TUs) generated, these new tools will be highly customizable and upgradable whenever new fluorescent proteins or promoters become available. Thus, in addition to producing a single-locus multi-hormone reporter, this project will also: A) popularize synthetic biology tools, such as the GoldenBraid1 (GB) gene assembly technology, among plant biologists; B) streamline rapid and quantitative pipeline to evaluate the function of individual genetic parts and modules; C) test the limit on the number of genes that can be routinely monitored simultaneously by ????????????????generic??????????????? labs not specialized in imaging techniques; D) explore new approaches that combine CRISPR-Cas9 and recombineering to stack multiple genes (up to 150 Kb) in a single TAC clone.

Title: Regulation of translation in tomato fruit ripening Rationale: Gene expression changes that accompany fruit ripening have been studied extensively at the transcriptional level, but little is known about ripening-associated shifts in the efficiency of transcript translation. The successful implementation of Ribo-seq in plants makes it now possible to monitor changes in transcript translation at a whole-genome scale and single-codon resolution. Recent studies by our and other groups that examined the effect of various stimuli on the efficiency of mRNA translation indicate that translational control is an important component of gene expression regulation in plants. Hypothesis: We hypothesize that fruit ripening is accompanied by changes not only in transcript abundance, but also in the rates of translation of a subset of mRNAs. The identification and mechanistic understanding of ripening-associated gene-specific translational shifts will open new avenues to developing novel approaches for the intelligent control of fruit ripening. Specific objectives: (1) Adapt our existing Ribo-seq protocols to tomato fruits. (2) Perform a time-course experiment to assess the role of translation regulation in fruit ripening. (3) Explore the potential of using specific regulatory cis-elements responsible for the translational repression in response to hormone ethylene to delay ripening-induced fruit softening. Approach: We will optimize conditions for polysomal RNA isolation and RNase digestion to enable Ribo-seq on developing fruits. Our prior experience with implementing and streamlining Ribo-seq in Arabidopsis and tomato seedlings will guide our efforts on adapting the protocols to tomato fruits rich in acids, sugars, and secondary metabolites by manipulating the extraction buffer composition and RNase digestion conditions. Ribo-seq and RNA-seq will be carried out on tomato fruits at 6 developmental stages (immature green, mature green, breaker, pink, red, senescing fruit) to identify transcripts that change in their translation efficiency during fruit development. Bioinformatic analysis will then be performed to find cis-elements enriched in translationally-regulated genes. We will also test the 3??????????????????UTRs of Arabidopsis and tomato EBF2 genes (that are required and sufficient for the translational inhibition of gene expression in the presence of the ripening hormone ethylene) for their ability to control the timing of fruit softening when fused to PECTATE LYASE (PCL). The fruits of PCL knockout and knockdown lines remain firm due to the reduced activity of the major fruit softening enzyme, PCL. Because ethylene evolution during fruit ripening peaks in pink fruits and ultimately declines in mature fruits, we expect that the fruits of pcl knockouts complemented by PCLp:PCL-3??????????????????EBF2 will remain firm until full fruit maturity. In pink fruits of these lines, high ethylene concentrations will block translation of the PCL mRNA via the 3??????????????????UTR of EBF2, whereas in mature red fruits translation of PCL will be resumed due to the relief of ethylene-mediated translation inhibition, and fruits will ultimately soften. Importantly, keeping pink fruits in ethylene during transportation and storage should block fruit softening until they reach consumers. Potential impact and expected outcome: Translational regulation of gene expression is an underexplored niche of science that, once better understood, will provide new ways to controlling gene activity in agriculturally important processes, in this case, fruit ripening. This high-risk, high-reward project, if successful, will not only generate the first map of the fruit ripening translatome and provide candidate cis-regulatory elements to be dissected in future studies, but also test the utility of previously identified regulatory regions that drive translational control in response to hormone ethylene for manipulating the timing of fruit softening without compromising other ripening-associated parameters, such as flavor and color development. This proof-of-concept study will serve as a fou

Since 2006, a disorder has been found in sweetpotatoes that causes internal discoloring and necrosis. Outbreaks of this disorder has occurred in specific businesses in North Carolina where the incidence has exceeded 50% while other growers may have only 2% or less occurrence. Complicating this problem, is that with this disorder no visible symptoms occur on the outside of the roots, making it impossible to detect by evaluating the outside of sweetpotato roots. Thus, roots with the disorder can reach the customer without detection. This has occurred with customers of grocery stores and restaurants after sweetpotatoes are cooked. Already some sweetpotato buyers have refused to buy Covington sweetpotato variety from North Carolina in fear of this disorder. We believe that the approximately $200 million dollar North Carolina sweetpotato industry is threatened by this disorder and an emergency situation currently exits. Continued shipment and not knowing the manner to prevent and/or understand the cause of internal necrosis could very negatively impact the North Carolina sweetpotato industry if not solved. This disorder has been studied since its appearance to find the cause(s) and develop remedies. However, despite much work, the cause of internal necrosis (IN) has not yet been determined. Three hypotheses have been developed after preliminary research was completed in 2012: combination of pre??A???a?sA???A?harvest and post??A???a?sA???A?harvest factors, ethylene, and disease. The aim of this proposal is to investigate these hypothesis to help understand the cause and ways to remedy the situation.

One of the main challenges of modern biology is to decipher the function of the thousands of genes that comprise an organism?s genome. Direct manipulation of DNA sequences in their native chromosomal context (e.g. addition of fluorescent tags to allow for the direct visualization of gene products or introduction of point mutations to evaluate the function of novel alleles) represents one of the most informative approaches used in gene function studies. Development of efficient genome editing strategies will not only greatly advance basic research, but will also benefit tremendously plant biotech industry by providing a more precise and less polemic alternative to the current transgenic approaches. The targeted alteration of a DNA sequence in its genomic context is typically achieved by means of homologous recombination (HR) in which the sequences present in a foreign DNA (the targeting cassette) direct integration of the cassette in a specific location in the recipient?s genome1. Not only does this approach represent the gold standard in many gene functional studies, but it can also be utilized to alter (increase or decrease) the activity of any gene. In spite of the tremendous power of HR-based approaches, their utilization has been limited to a handful of model organisms in which the rates of sequence-driven integration events of foreign DNA are sufficiently high. Unfortunately, this is not the case in most plants, including all economically important crops, where the frequency of random integration events exceeds by several orders of magnitude that of the homology-based integration. Thus, to bring the theoretical advantages of HR to a practical arena, a dramatic increase in the rate of HR needs to be achieved. So far, two main approaches have been exploited to boost HR efficiency. On the one hand, overexpression of genes involved in homologous recombination in heterologous systems has been employed, e.g. ectopic expression of RAD54, the yeast chromatin remodeling gene required for high levels of HR in this species, has been shown to increase the frequency of HR in Arabidopsis 20 to 30 fold2. Despite this significant improvement, the RAD54 strategy still requires the generation and characterization of hundreds of transgenic plants in order to identify a single HR event, making this approach unpractical. Other recombination systems, such as the λ RED3, have also been shown to result in a tremendous boost of HR rates in bacteria, but thus far have not been tested in plants. The second main strategy to increase the frequency of HR is based on the observation that the levels of HR increase at the chromosomal site where a DNA break has occurred4,5. Recent developments in the engineering of zing-finger6, TALEN7 and, more recently, Cas9 nucleases8 make it possible to create such chromosomal breaks at preselected locations. Of the four types of nucleases, Cas9 appears to be the most promising due to the tremendous simplicity and flexibility of the Cas9 system 9, but its practicality in plants remains to be demonstrated.

Summary. Interaction between hormonal and developmental signals is emerging as a critical mechanism in the generation of the tremendous phenotypic plasticity characteristic of plants. A good example is response to ethylene that depends on tissue type, developmental stage, and environmental conditions. Characterization of the Arabidopsis wei8/taa1 mutant has uncovered a small family of genes that mediates tissue-specific responses to ethylene. Biochemical studies indicate that TAA1 is a long-anticipated tryptophan aminotransferase in the essential, yet genetically uncharacterized, indole-3-pyruvic acid (IPA) branch of the auxin biosynthetic pathway. This breakthrough has opened new avenues for examining fundamental questions in auxin biology and, therefore, paved the road for future hormone interaction studies. In this proposal, the following three major aspects of the auxin biology will be examined: 1) The biological significance of local auxin biosynthesis in several well-defined developmental processes and in response to ethylene will be investigated. Our results from the characterization of TAA1 and TARs challenge the longstanding paradigm that "local" auxin biosynthesis plays no direct role in the generation/maintenance of the auxin gradients essential for development and responses to the environment. An integrative approach that takes advantage of the latest genetic and cell biology tools will be used to address this question. 2) The relationship between the TAA1 and the YUC routes of auxin productions will be examined. The comparison of expression patterns and mutant phenotypes between the two best-characterized auxin biosynthetic gene families, TAA1/TAR and YUC, defies the established model of two independent auxin production routes. Genetic and biochemical approaches will be employed to resolve this controversy. 3) Genes acting downstream of TAA1 in the IPA route of auxin biosynthesis will be sought out. Molecular genetic approaches in both Arabidopsis and the auxin producing bacteria Azospirillum brasilense will be utilized to identify key genes in this novel plant auxin biosynthetic pathway.