Jose Alonso Bellver

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.

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.

Auxin is an essential plant hormone that participates in the regulation of nearly every aspect of plant life cycle, from embryo development to meristem maintenance, and from defense against pathogens to shade avoidance. Despite the key role of auxin, the biosynthetic pathways that plants utilize to produce this hormone are largely unknown. In fact, none of the several proposed routes of auxin biosynthesis have been elucidated in their entirety and, in most cases, just a single gene of a multistep pathway has been identified. Thus, for example, the IAM pathway is defined by the AtAMI1 gene, the IPyA pathway is defined by the TAA1 gene family (and now also the YUC family), etc. Despite major gaps in our understanding of auxin synthesis in plants, it is firmly established that indole-3-acetic acid (IAA), the prevalent form of auxin in plants, can be produced from the amino acid tryptophan (Trp) or from the Trp biosynthetic intermediate indole-3-glycerol phosphate. Trp, on the other hand, in addition to serving as a precursor in the IAA biosynthesis, is also an essential building block for proteins, as well as the precursor for a number of defense compounds such as indole glucosinolates and camalexins. Trp itself is synthesized via the shikimate pathway along with two other aromatic amino acids, phenylalanine (Phe) and tyrosine (Tyr). Phe and Tyr, like Trp, serve as the precursors to a large array of secondary metabolites including anthocyanins, flavonols, lignins, etc. Thus, one can imagine the auxin biosynthetic pathway as one of the many final branches of the large metabolic tree of the shikimate pathway. This raises another important question about auxin biosynthesis, i.e. how do plants coordinate the activity of all these different metabolic routes that feed on common precursors? Supposedly, this is achieved by a refined (and yet unknown) mechanism that coordinates the activity of the different metabolic branches that comprise the shikimate pathway and that operates at the cellular level. The current proposal will focus on addressing two open questions in auxin biology. What are the genes that compose the different auxin biosynthetic routes? And, how are the auxin biosynthetic pathways coordinated within the large metabolic network in which they are embedded? To address these key questions, we will focus on the following two objectives. (1) We will systematically examine the proposed indole-3-pyruvic acid (IPA) independent routes of auxin biosynthesis. Several genes previously implicated in key steps of these routes will be characterized using a combination of genetic and biochemical approaches to re-evaluate their role in auxin biosynthesis. Furthermore, to shed light on the unknown components of the IPA-independent routes of auxin production, novel genetic and chemical screens will be conducted. (2) We will also define, at cellular resolution, the regulatory network responsible for the coordinated activity of the different branches of the shikimate pathway. This will be achieved by monitoring the spatial and temporal expression of a set of 84 genes from selected branches of the shikimate pathway. Perturbation of the system using pharmacological means will be used to identify the interconnection between the different components of the network.

Project Summary Intellectual Merit This proposal requests support for the 24th International Conference on Arabidopsis Research (ICAR) to be held at the Convention and Exhibition Centre in Sydney, Australia, June 25-28 2013. The majority of breakthroughs in plant science in the last 20+ years have relied on development of Arabidopsis thaliana as a reference for both research and international collaboration. Thousands of researchers around the world use Arabidopsis in their studies; knowledge derived from such research informs all aspects of basic plant biology, due to the unique features of this model organism that rapidly enable new discoveries. Critically, paradigms established using Arabidopsis have, and will continue, to be applied to crop species, thus paving the way for rational improvements in a variety of agricultural traits. The success of this research field has been greatly facilitated by the openness and collegiality of the community fostered through multiple international forums including ICAR, which brings together approximately 1,000 participants to exchange scientific results and report on progress in the field. The conference will cover a broad range of important topics including Evolution and Natural Variation; Small RNAs, RNA and Epigenetics; Transgenerational Inheritance; Development; Hormones; Cell and Organelle Biology; Intracellular Signaling; Cell to Cell Communication; Abiotic Stress; Biotic Stress/Interactions; Energy Biology/Metabolism; Photosynthesis and Water; Phenomics; Proteins and Posttranslational Regulation; Emerging Technologies and Systems Biology; Emerging Topics and What's Hot, and Translational Biology. There will also be a series of satellite meetings on plant energy biology, epigenetics and high-throughput plant phenomics. The meeting includes a special tribute in memory of Simon Chan, a highly talented early-career U.S. scientist who tragically passed away in 2012. The ?Simon Chan Symposium? will feature presentations in the research area in which he performed his groundbreaking studies, notably, by demonstrating the practical feasibility of a ?reverse breeding?, one of the most sought goals of plant breeding, in Arabidopsis. Importantly, ICARs have proven to be an extremely effective venue for exposing young scientists to the field and for encouraging interactions between younger and more senior researchers. In addition to platform talks the conference will include 36 speakers chosen from submitted abstracts with an emphasis on presentations by students, postdoctoral researchers, and faculty members at early stages of their careers. There will also be community-organized workshops that allow additional speakers to present their research. This will ensure presentation of the latest results and provide important career development for young scientists. Broader Impacts The ICARs, which provide the primary annual opportunity for scientists in the Arabidopsis community, as well as other plant biologists to meet and share the latest research, are a key component in the continuing success of the worldwide Arabidopsis community. ICARs have proven to be a highly effective venue for enhancing information exchange, creating new networks and establishing new collaborations. The ICARs are also critical to facilitate higher-level organization of the plant research community by providing a venue for groups like the International Arabidopsis Informatics Consortium (IAIC), the Multinational Arabidopsis Steering Committee (MASC), and the North American Arabidopsis Steering Committee (NAASC) to convene annually. These meetings allow discussions of the current status of the international Arabidopsis community as well as development of future plans and research directions. Ensuring the participation of scientists from diverse backgrounds is critical to the vitality of science in the U.S. A key goal is to increase participation among under-represented minority scientists and early-career scientists. To this end we will fully support participation by under-represented minority scientist

ARI-R2: Renovation of the North Carolina State University Phytotron for Improved Environmental Control and BSL-3 Containment. North Carolina State University (NCSU) faculty and students have a compelling need for a state-of-the art controlled, environmental facility (Phytotron) with containment for investigation of high-risk plant pathogens and other microbes. The four-story Phytotron was built in 1968 with most of the construction (~$1.5M) and first eight years of operating funds provided by the NSF, and 33 years of continued maintenance and staffing provided by NCSU. After 41 years however, the NCSU Phytotron is in dire need of upgrades to meet present and growing demands for controlled environmental research facilities for plants, insects and small mammals. This application proposes critical infrastructural upgrades to (1) renovate the core environmental system by replacing chillers and pumps, connecting to university chilled water lines, re-insulating chilled water and glycol lines, and applying epoxy coating to chamber and greenhouse floors; (2) replace controllers that modulate the environmental conditions for individual environmental chambers, and modernize and increase the precision of CO2 controls; and (3) convert a plant dark room and greenhouse into a BLS-3 facility for investigations with highest risk viral, bacterial, fungal and nematode plant pathogens and select agents. Intellectual Merit. Biologists in the post genome era face a major challenge in determining the function of the thousands of identified genes, many of which are under environmental control. Often, the phenotype of a mutant and the expression pattern of responsible gene(s) can be properly defined only in precise environmental conditions. Renovation of the Phytotron will provide researchers with the large-scale controlled environment capacity to take full advantage of publically accessible genetic tools not only to accelerate information-rich phenotyping in model plants but to translate the same high-throughput screens to crops, wild relatives and non-native species. Control over CO2 concentration will open new opportunities for NCSU researchers involved in global change research and predicting the responses of individual organisms and ecosystems to environmental change. These demands, as well as the growing need for studies of phenotypic plasticity needing reproducible combinations of climatic factors and ecological studies that require the capacity to simulate a variety of environments, make this facility an essential resource for future, competitively funded investigations. Similarly, upgrading a designated area of the facility for BSL-3 containment will provide a research environment and foster international collaborations to better understand the fundamental biology of plant pathogens that already cause severe disease problems in subtropical/tropical regions and pose an increasing threat to the U. S. and global food supply chain. Broader Impacts. The NCSU Phytotron is an outstanding testament to the value of investing in research infrastructure. This NSF-funded legacy is a valuable University-wide facility and focal point for training and research at all levels. The infrastructure renovations proposed here will allow this unprecedented resource to continue to fulfill this mission by becoming a high-throughput public facility for phenotypic analyses of many different plants. Graduate and undergraduate students from NCSU, summer REU programs involving students from non-Research I universities and other outreach programs for underrepresented groups use the Phytotron for independent research projects under the direction of a diverse representation of faculty who serve as role models and mentors. The renovations will have immediate scientific impacts as NCSU researchers can acquire pathogens and microbes from collaborators around the world by having a centralized facility inspected and known to meet or exceed containment guidelines. Unemployment in the State of North Carolina is over 11% and above the national average.

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.

The main objective of this proposal is to develop the analytical tools to study an essential, yet still poorly understood, step in gene expression: mRNA translation. While other critical aspects of gene activity, such as changes in the level of a mRNA or a small regulatory RNA, can be easily examined and precisely quantified, only recently it has become technically possible to obtain the same type and quality of information on changes in translation. These new technical developments have opened a new window of opportunity to advance our understanding of key biological processes. Thus, the implementation of this new technology will provide a new competitive edge to a broad community of researchers at NC State University interested in the regulation of gene expression. The complementary research interests of the PIs, their proven experience in employing these types of technologies, and the qualities of the chosen experimental system, provide strong guaranties of the success of this proposal.

PROJECT SUMMARY. Senior Personnel Jose M. Alonso. North Carolina State University (PI). Summary of the Proposed Project Towards the ultimate goal of deciphering the function of every gene in the Arabidopsis genome, the 2010 program recognizes as paramount the generation of high-resolution spatial and temporal maps of gene expression patterns and sub-cellular localization of the corresponding gene products. Translational fusions between a reporter gene, such as GFP, and a gene of interest (GOI) in its chromosomal context provide the most accurate information on that gene?s expression pattern. In the absence of efficient homologous recombination system in Arabidopsis, large Arabidopsis artificial chromosomes can be precisely tagged with GFP by homologous recombination in E. coli and then transferred to the Arabidopsis genome. Before embarking on a large and expensive whole-genome project, the efficiency and effectiveness of the proposed approach will be tested on a selected set of 200 genes. By including in this gene list a group of genes with well-characterized expression patterns (25), as well as genes with well-defined mutant phenotypes, the accuracy of the obtained information and, therefore, the utility of the system will be evaluated. The efficiency of each step in the process and the ?scalability? to a whole-genome level will be examined. Finally, the utility of using additional tags (IREScp-GAL4, in particular) will be investigated, exploring the potential of employing the generated tagged clones for other purposes. For example, the GOI-IREScp-GAL4 lines obtained could be used to confer specific well-defined expression patterns to any gene of interest and to test the functional relevance of a particular expression pattern.