Anna N Stepanova

Assistant Professor of Plant and Microbial

Biology

PhD, University of Pennsylvania

Postdoctoral, North Carolina State University

Jose M Alonso

Professor of Plant and Microbial Biology

PhD, Universitat de Valencia, Spain

Postdoctoral, University of Pennsylvania and The Salk Institute for Biological Studies

                                     General interests

    Our main interest is to understand the molecular circuits plants use to integrate environmental and developmental signals to produce specific responses. Towards this general goal we have been focusing on the identification of the molecular “signal integrators” or “logic gates” involved in the interaction between two plant hormones, ethylene and auxin, in the regulation of root growth. Using a multidisciplinary approach (genetics, molecular biology, genomics, metabolomics, cell biology, etc.), we have uncovered a complex multistep integration process with both spatial and temporal components. Our research has shown that ethylene activates the transcription of auxin biosynthetic genes in the root meristem (root tip) and then auxin is transported upwards to where it sensitizes the cells in the division zone enabling them to properly respond to ethylene. Our more recent findings suggest that translation regulation represents a key aspect of this “sensitization” mechanism triggered by auxin. In parallel, these studies have allowed us to decipher the first complete auxin biosynthetic pathway in plants and we continue to investigate the role of auxin biosynthesis in development. Finally, we combine our interests in basic biology with the development and implementation of new genetic technologies to accelerate discoveries in plant biology. Currently, we are working on three main areas: gene modification in a chromosomal context using recombineering approaches, high-resolution whole-genome analysis of translation using next-generation-sequencing (NGS) enabled ribosome footprinting, and the implementation of synthetic biology approaches to generate transcriptional reporters for plant hormones.


Auxin Biosynthesis

  
Plants, as animals, produce a set of “communication” chemicals called hormones. These small messengers are made in very small quantities as even a few of such molecules are capable of triggering dramatic changes in growth and physiology. In plants, one of the most studied hormones is auxin. The powerful effects of this hormone have been known for a long time. In fact, we now know that auxin was the mysterious “influence” transmitted from the tip of the growing plant in the famous experiments of Charles Darwin and his son on how plants bend towards the light. In addition to the role of auxin in phototropism (bending of the plants towards the light), this growth regulator plays a critical role in many other processes in plants, some of which are of great agricultural interest. Thus, for example, accumulation of this hormone in select parts of the plant (specific cells or group of cells) provides the critical information needed to decide when, where and how critically important organs such as flowers, fruits and seeds should be made. In the past 20 years, scientists have uncovered many of the mechanisms by which auxin triggers some of these plant responses. However, only recently we have started to uncover how plants make this essential hormone. In the last 5-10 years, we and others have discovered what seems to be the main route by which plants produce this hormone, what we call the IPyA route. With this discovery new challenges emerged. One such critical challenge is how the biosynthesis, transport and response to this hormone work together to control plant’s functions. To start to address this question, we have generated a large collection of genetic tools that allow for the direct monitoring of the activity of over 100 genes involved in the production, transport and response to this hormone with cellular resolution. The information provided by this new comprehensive tool kit will be essential to creating a much needed global view of this hormone’s activity and how biosynthesis, transport and response interact with each other to achieve both the precision and resilience needed to generate extremely complex structures such as flowers and fruits.  

An additional question that emerged from the discovery of the main auxin biosynthetic pathway is whether or not other previously proposed routes of auxin production are truly functional in plants. Systematic scrutiny of this important question using different experimental approaches suggests that none of the genes previously proposed to function in making auxin through this alternative routes have a significant contribution to the production of this hormone under the studied conditions. Since we know that plants are capable of making this hormone by means other than the well-established IPyA route, how plants make this hormone through this other route(s) remains an important open question in hormone biology.  


Translational regulation of hormone responses (Visit out Plant-Ribo-Prints website)

    Plants, as sessile organisms, need to constantly adjust their intrinsic programs of growth and development to the environmental conditions. This adaptation often involves changes in the developmentally predefined patterns of activity of one or more phytohormones. In turn, these hormonal fluctuations lead to alterations at the gene expression level and to the concurrent changes of the cellular activities. In general, the hormone-mediated regulation of plant development is 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 novel regulatory elements indicates that this regulation of translation is not miRNA dependent, and that the element
identified is also responsive to the plant hormone auxin, implicating this element in the interaction between ethylene and auxin. 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 for identifying, characterizing and utilizing additional regulatory modules of gene expression in plant species of economic importance. Towards that general goal, a plant-optimized ribosome footprinting methodology is being 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 is maximizing the detection sensitivity and diversity (early vs. late activation) of translational regulatory elements. The large amount and dynamic nature of the generated data will be also utilized to generate hierarchical transcriptional and translational interaction network models for the two hormones and to explore the possible use of these diverse types of information to identify key regulatory nodes.  Finally, the comparison between two plant species is providing critical information on the conservation of the regulatory elements identified and, thus, informs research on future practical applications.


Synthetic biology approaches to generating the first plant multi-hormone reporter (Hormometer)

    Both plants and animals, like humans, use a set of hormones to control growth and development.  These hormones are small chemicals produced by the plants or animals in extremely minute amounts, but capable of triggering dramatic changes in the organism. All multicellular organisms can produce and carefully distribute these growth regulators to ensure proper development and defense against harmful environmental conditions or infections. In plants, there are nine classes of plant-produced chemical hormones that govern plant growth. These control a wide range of developmental processes, from seed germination to fruit ripening.  Despite the critical role of plant hormones, they are somewhat difficult to study, in part because it is challenging to know specifically when and where the hormones are made and act. One of the ways the activity of the hormones can be visualized is with the help of the so-called fluorescent reporter genes that make parts of the plan
ts glow under UV light if the hormone is active in that part of the plant. We are producing a set of synthetic reporter genes that will make it possible to visualize the activity of multiple hormones at once, all in a single plant. Importantly, these reporters will be useful for the analysis of hormones in many plant species, including crop and ornamental plants and even trees.

    A major limitation of existing hormone reporters is their inability to monitor multiple hormones in a single plant line.  Currently, the best available synthetic reporters (e.g., DR5:GUS, DR5v2-GFP, or DII-Venus for auxin and EBS:GUS for ethylene) need to be crossed or transformed into the background of interest (e.g., one's favorite mutant) individually due to the presence of the same selectable markers, identical reporter genes, and/or major silencing issues caused by the repetitive use of the same or overlapping DNA elements.  Multi-gene hormonal reporter constructs and transgenic lines would greatly facilitate the multi-faceted phenotypic analysis of pleiotropic mutants, as well as of the effects of stresses and other treatments that typically alter several hormonal pathways in parallel. For example, it is easy to imagine the benefits of simultaneous monitoring of the dynamics of all major hormones’ activities at cellular resolution to understand processes as important as drought, nutrient deficiency, plant-microbiome interactions, or hormone crosstalk.




Outreach

    Our Plants4Kids outreach activity is targeted at school-age kids (2nd to 5th grade) and has been running since 2011. This is a bilingual (English and Spanish) web-based science dissemination program that consists of a set of 14 experimental modules in plant biology designed specifically for kids. Each experimental module is divided into six sections: the question that will be addressed in the experiment, the materials that will be needed, the directions on how to perform the experiment, helpful suggestions, expected results, and follow-up questions. By providing step-by-step bilingual directions for performing easy and inexpensive (and, most of the time, free) experiments, we hope to include children coming from low-income families that often have very limited resources and/or poor English language skills. To promote our outreach program, every second Saturday of the month we run a hands-on booth at the North Carolina Museum of Natural Sciences, as well as occasional demos
at local elementary schools. Since the inception of Plants4Kids in 2011, our program has reached thousands of kids over the course of over 70 live demos (ranging in duration from 2 to 8 hours each). The goal of these activities is to stimulate interest in science among young people and to make basic science accessible to the underrepresented Hispanic community.

    Encouraged by the success of our Plants4Kids we have now started a new outreach activity targeted to older kids, Plants4Teens. The main goal of this outreach activity is to promote interest among middle- and high-school-age students in combining biology, computers, and engineering. We are trying to achieve this by means of simple animated videos that explain basic concepts in biology and its interface with other disciplines.




 

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