Federica Brandizzi - US grants

Intellectual merit. It is well established that cells of plants, fungi, and animals contain organelles that carry out vital functions for the life of a cell. What is yet to be learned is how the identity of organelles is established and maintained. In this project, the plant Golgi apparatus will be used as a model to address this fundamental question. In plants, the Golgi apparatus is composed of highly mobile stacks of membranes that are dispersed on tubules of another organelle, the endoplasmic reticulum (ER). The plant Golgi receives proteins and membranes from proximal and distal locations; it is involved in their modification and sorting to their final destinations and in synthesizing the cell wall that encapsulates plant cells. The flow of membrane proteins through the Golgi as well as the dynamic exchange of peripherally associated proteins require continuous remodeling of this organelle. Yet, the stacks withstand dispersion during the intense traffic of proteins to and from the Golgi. This raises the fundamental biological question: How does the Golgi retain its integrity (i.e. architecture and composition)? The integrity of the Golgi depends on the organelle's ability to dynamically maintain its steady-state architecture and membrane distribution during protein traffic. Using a screen specifically developed for the plant Golgi in the Brandizzi laboratory, a unique set of mutants was identified in which an established Golgi marker is partially mis-targeted to other organelles. This screen is exciting because it sets the foundation for identifying key players of the machinery that controls plant Golgi integrity. In this project, characterization of the Golgi mutants will be carried out to identify the protein(s) responsible for the maintenance of the architecture and membrane steady-state distribution at the plant Golgi. This work will lead to answers to fundamental questions regarding organelle biogenesis and important differences in Golgi organization and function across eukaryotic biological systems.

Broader impacts. Plants are the primary energy source of all biospheric development: all animals survive on plants, both directly and indirectly. The secretory pathway of plants plays a fundamental role in the conversion of fixed carbon into energy-rich materials such as proteins, lipids, and complex sugars. These plant-derived products are important not only for nutrition, but they have the potential to be used as renewable fuels, lubricants, textiles, and building materials of all kinds. The plant Golgi is a key organelle in cell wall biosynthesis and storage protein deposition. Because unique variations exist among systems as a result of evolutionary adaptation, it is important to study the plant Golgi if humanity is to tap into the full potential of plants as primary providers of bio-molecules on earth. This project will also enable the Brandizzi lab to continue their engagement in teaching and outreach activities to communicate to students and teachers the scientific discoveries in plant science and their impact on our society.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

The study of plant biology has advanced our understanding in areas not only related to agriculture and food quality, but also in the areas of human biology. Recent advances in technologies in the area of biochemistry, cell biology and genetics have enabled plant scientists to address biological questions previously unattainable. This award provides support for the acquisition of a laser capture microdissection (LCM) system and ancillary equipment for advanced histologic analysis of plant materials at Michigan State University. LCM is an advanced method for analyzing single cells in living multicellular organisms. Using this technique, cells from a target microscopic region can be selected, isolated, and analyzed. In short, this yields precise cellular snapshots of cell diversity and the mechanisms controlling processes as varied as development, stress, metabolism, pathogenesis, and senescence. As a world leader in the experimental plant sciences, MSU is extremely well placed to use and disseminate this powerful technology, and the addition of LCM will leverage existing technological strengths at MSU to catalyze breakthroughs in plant biology. In addition, through hands-on training and education, post-graduate, graduate, undergraduate and even high school students will receive specialized training using LCM, as well as downstream genomic and proteomic techniques at MSU.

? DESCRIPTION (provided by applicant): Protein synthesis in the endoplasmic reticulum (ER) can be dramatically upregulated under stress conditions. To avoid jamming of protein translocation and folding, the ER senses early indicators of such stress conditions and responds with the so-called unfolded protein response (UPR). This is an ancient signal transduction pathway that leads to the rapid replenishment of ER chaperones and other folding factors and avoids energy- requiring repair mechanisms before committing to apoptosis. The UPR is critical to sustain cell growth and development and to combat disease and abiotic stress. Interestingly, the efficacy of the UPR varies largely among individuals of the same species, but the underlying molecular causes are unknown. To initiate the UPR, yeast relies heavily on the action of a conserved ER stress sensor, Ire1p. During the course of evolution, the suite of UPR sensors has expanded to accommodate more specific responses in a multicellular context. The basic activation mechanisms and general function of the ER stress sensors are largely known from in vitro studies and cell culture analyses. However, how the UPR regulators work coordinately to sustain healthy cell growth and development with a minimum of energy costs is unknown. We wish to address this fundamental question in Arabidopsis thaliana, because of the conservation of plant and metazoan UPRs, the vast availability of genetic diversity and genomics resources for this model species, and the relevance of the plant kingdom as a source for renewable energy, food, and materials. The immediate goals of this proposal are 1) to define the molecular determinants underlying intra-specific UPR variability using natural populations with broad genetic diversity, 2) to understand the mechanisms that control homeostasis among the various UPR pathways in vivo, and 3) to define non-conventional mechanisms that modulate ER stress responses in intact organisms. To achieve our goals, we will pursue our genome-wide studies to characterize UPR diversity as well as our functional genomics analyses to define the mechanisms for homeostasis of the UPR signaling pathways in vivo. We will also use advanced next-generation sequencing strategies to define post-transcriptional modulation of the UPR. Our results will lead to a broad and deep understanding of the complexity of the UPR signaling network during ER stress in the context of complex multicellular organisms. Adding plants as an evolutionarily distinct and tractable model for the study of the UPR in complex organisms is also important because it will allow comparing and contrasting plant, yeast, and animal UPRs, and thus will provide significant insights into these systems, adding to the fundamental knowledge of eukaryotic cell biology at large. Our results will not only enhance our understanding of human growth and disease, they will also permit the development of drugs in a tractable multicellular model, and contribute to our knowledge of limiting factors in agricultural processes and plant biotechnology designed to sustain food security on earth.

INTELLECTUAL MERIT
A central question in eukaryotic cell biology is how the identity of organelles is established and maintained. The endoplasmic reticulum (ER) is an essential organelle of the secretory pathway for the production of a wide variety of the cell's building blocks, as well as for the control of essential stress and hormonal signaling pathways. To achieve maximum efficiency, the ER assumes a unique architecture characterized by a network of interconnected membrane tubules and sheets to form closed polygons. Discoveries in ER integrity are emerging from studies in various model organisms/systems such as fruit flies, yeast and cultured human cells. Compared with these systems, however, the ER has acquired unique morphological and functional features in the plant lineage that are likely linked to its important role in lipid synthesis together with chloroplast, intercellular communication, protein storage and plant-specific hormone signaling. The goal of this project is to investigate the unique regulatory mechanisms that maintain plant ER integrity by using genetic mutants, live cell imaging and biochemical approaches. These approaches have identified several genes that encode critical players involved in ER integrity and architecture including an Arabidopsis ER-associated dynamin-like protein, named RHD3. The project research aims to elucidate the mechanistic role of RHD3, as well as other gene products identified by mutant screens, in maintaining the architecture and functions of the plant ER. Since integrity and function are two inextricably linked features of the ER, the research will advance the general understanding of ER roles in the plant secretory pathway in the context of a multicellular organism and contribute to answering fundamental questions regarding differences in ER organization and function across eukaryotic systems.

BROADER IMPACTS
Plants are the direct or indirect primary carbon and nitrogen source of all animals and humans, in addition to their role in providing materials and fuels. The secretory pathway of plants plays a fundamental role in the conversion of fixed carbon into energy-rich materials, such as proteins, lipids and complex sugars. These plant-derived products are not only important for nutrition, but have the potential to be used as renewable fuels, lubricants, textiles and building materials. Because unique variations exist among eukaryotes as a result of evolutionary adaptation, it is important to study the unique properties of the plant ER which is the key organelle for the biosynthesis of important building blocks of cells and for essential signaling path-ways in growth, development and stress responses. The project will also broaden the impact of on the plant cell science research community by providing unique plant lines and constructs which will be made available to other plant cell biologists. The research will promote teaching, research training and outreach activities through multiple avenues both at Michigan State University as well as the local community. In particular, project personnel will continue to communicate to students and teachers discoveries in plant science and their impact on the society by engaging students and teachers in research activities in the lab and by performing science presentations at schools.

This research will investigate strategies for protecting maize, the major U.S. food, feed and fuel crop, from environmental stress. Maize can perceive and respond to adverse environmental conditions through a process called the unfolded protein response (UPR). Folding is a critical, but delicate step in the biosynthesis of proteins, and it can be easily upset by adverse conditions, such as high temperature. When this happens, misfolded proteins accumulate and activate a cascade of stress response genes. One of the objects of this project is to characterize in depth the cascade of genes to better understand how they protect maize from stress. Investigations in other plants has revealed that stress conditions eliciting the UPR also activate a process called autophagy in which plant cells repair stress damage. Therefore, another aim of this project is to discover how stress signals activate the autophagy machinery. In response to stress, other plants slow down protein synthesis to prevent overburdening the process of protein folding. In this project an investigation will be conducted to determine whether maize does this by degrading some of the messenger RNAs encoding proteins. Finally, attempts will be made to modify the UPR and probe more deeply into its operation by using new gene editing techniques. In parallel, there will be a focus on links between learning and research at the undergraduate level. This will be done through the development of easily accessible training on line and peer-support learning communities.

In this project, it is expected that new gene targets involved in ER stress responses in maize will be revealed through extensive transcriptomic analysis. It is anticipated that those targets may provide a clearer picture of both cell survival activities and cell death mechanisms in response to stress. In addition, these analyses will aid in uncovering the signaling pathways by which stress elicits the proliferation of the ER and activation of autophagy. While some UPR responses involve the upregulation of stress response genes, other responses result from the degradation of specific RNA transcripts brought about by Regulated IRE1-Dependent RNA Degradation (RIDD)and microRNA action. In this regard, the degradome as well as the transcriptome will be used to determine the role of selective RNA transcript degradation in maize stress responses. This research project is also expected to reveal through 'translateome' analysis whether ER stress is also mitigated in maize by selective and/or global regulation of the translation of RNA transcripts. A compelling reason for the selection of maize as a model for these studies is that the UPR has already been demonstrated in maize in the field. Therefore, variation in stress response in different lines of maize will be studied both in the laboratory and in the field with the goal of identifying genetic determinants that condition the UPR.

In addition to providing commodity materials and fuels, plant cells are an essential source of oxygen and nutrients to humans. Like all eukaryotes, the life of plants depends on the function of specialized structures, known as organelles that populate the subcellular environment. An essential organelle, whose function and morphology are largely conserved across eukaryotes, is the endoplasmic reticulum (ER). This project seeks to understand how the ER establishes and maintains its functional and morphological identity. As the ER is at the core of the cell's biosynthetic machinery, and because significant variation in ER biology exists among eukaryotes, studying the ER within model plant systems provides an essential foundation to improve plants as primary providers of useful biomolecules and to support life on this planet. This project on the Arabidopsis ER will also provide new fundamental knowledge that will influence the study of other organisms that share mechanisms underlying ER biogenesis with plants. In addition, it will provide societal benefits through education of the next generation of scientists, engagement of high-school students and teachers in research, and by communicating discoveries in plant science and their impact on the society through outreach activities.

A central question in cell biology is how organelles establish and maintain their identity. Due to its rich, mutable morphology, the ER is a wonderful model to explore this question. The ER adopts an intricate web-like architecture via cytoskeleton-driven remodelling and anchoring of subdomains to the plasma membrane (PM). However, the mechanisms underlying ER shape as well as differentiation and function of ER-PM subdomains are largely unknown, especially in plants. The research will capitalize on the discovery of novel components of the Arabidopsis proteome that control ER morphology and interaction with heterotypic membranes, including the PM. Using a suite of advanced live-cell imaging approaches, functional genomics analyses and mathematical modeling, the project will characterize a novel plant ER shaper, define the molecular composition of the ER subdomains interacting with the PM and determining how such subdomains influence the biosynthesis and deposition of the extracellular matrix. The results will help create a new paradigm of plant ER structure and function. Since ER integrity and function are inextricably linked and the ER presents species-specific features, this research promises to significantly advance basic understanding of plant ER architecture and function, and provide a framework for comparative insights into ER biology among eukaryotes.

This project develops new mathematical models of the interactions of multicomponent lipid membranes with oils and proteins. Lipid membranes are fundamental building blocks of cellular structure forming the separating walls, the plumbing, and the assembly points for many proteins. The work will be conducted by an interdisciplinary team of mathematicians and plant biologists and will focus on validating experimental observations of lipid droplets against predictions of the multicomponent lipid models. Lipid droplets are of significance to public health for their role as a signal of the onset of metabolic syndrome, which has a correlation with the development of small fatty inclusions (lipid droplets) within the liver. Lipid droplets are being pursed in oil seeds and algae as energy storage compartments to improve the energy content of the biomass. The mathematical elements of the proposal identify key bottlenecks within the rearrangement of lipid structures with the creation and destruction of minimal energy pathways within a higher dimensional system, these pathways correspond to optimal packings of lipids and oil that contribute to the observed self-assembled structures.

The project presents a detailed analysis of the structure of the free energy governing the low energy connections that represent amphiphilic morphologies and high energy barriers that are the minimal cost of reorganization. The PI and his collaborators investigate the central thesis that the dominant manifestation of the entropy of packing of amphiphilic molecules lies in the structure of this connection problem. Packing-based energy landscapes are strongly nonlinear, and fundamentally distinct from the more weakly nonlinear mixture-based models built upon the Cahn Hilliard framework. The packing-based energy will be validated against in-vitro experiments on lipid droplets, the prototypical example of a two-fluid amphiphilic system driven by interfacial competition, and are compared with entropic descriptions derived from short- chain limits of self-consistent mean field theory models. Specifically, the proposal uses spatial dynamics and singular perturbation techniques to develop robust pearling inhibition that stabilizes the pearling modes of connections, and tunes the existence and persistence of connections and barriers within factored Melnikov structure via piecewise defined and singularly perturbed dynamical system approaches. The two fluid model of lipid droplet formation will be used to investigate pinch-off and merging events while one fluid models address phase separation (rafting) and localized pearling. The models will be validated by comparison to entropic effects extracted from self-consistent mean field models of amphiphilic blends, to in-vitro experimental morphology of lipid droplets, and to the bifurcation diagram for castings of synthetic amphiphilic polymers.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The meeting 'IX Cell Wall Research Conference 2022 (IXCWRC): Cell wall research for new fundamental discoveries and applications in plant biology' will be held in East Lansing, Michigan on June 13-17, 2022. The meeting will focus on the plant cell wall, which makes up most of the plant biomass, interfaces with the external environment, and supports the growth of the entire organism. Understanding the cellular and extracellular processes necessary to build and maintain the plant cell wall is important because cell wall biosynthesis and remodeling during growth and disease are largely uncharacterized processes, and because of the relevance of the cell wall for food, fiber, fuel, and other materials that are indispensable to humans. A better understanding of how the cell wall is synthesized, deposited, and maintained during plant growth and in response to the environment can inform and improve biotechnological processes for increasing plant yield and resilience. Acquiring such knowledge requires the exchange of new ideas and results as well as the development and application of new technologies.

The IXCWRC aims to provide an open forum to discuss new ideas and theories, develop collaborations for scientists across the globe in a diverse and supportive environment, in addition to promoting and training the next generation of scientists through tailored workshops and activities. The NSF award will be used to develop an exciting program, particularly by broadening the participation of early career investigators to the meeting, facilitating their networking and fostering the foundations of an inclusive community in science.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.