Sheng Luan - US grants

The long term objective of the research projects in this proposal is to understand the molecular basis of signal transduction pathways in higher plants. Intra- and intercellular signal transductions are key processes that regulate cell growth, division and differentiation. Recent studies using natural products as probes have revealed many new signaling events in mammalian cells. One family of such natural products are immunosuppressive drugs cyclosporin A (CsA), FK5O6 and rapamycin. These drugs bind to their cellular receptors (referred to as immunophilins) and form receptor-ligand complexes that in turn target signaling molecules required for T cell activation therefore suppressing the immune response. In the absence of the drugs, immunophilins have been implicated in various cellular processes including protein folding and trafficking in both animal and yeast systems. In higher plant cells, as in T cells, certain signaling pathways are interrupted by immunosuppressive drugs. These pathways, unlike those in T cells, are initiated by plant hormones and modulate ion channel activities that control stomatal opening. In addition, plants produce a unique set of immunophilins that are localized in various subcellular compartments. In particular, two of the immunophilins are specifically expressed in green tissues and are localized in the chloroplast. These findings have uncovered a new family of proteins from higher plants and opened new possibilities of using immunosuppressive drugs as powerful probes to study intracellular signaling in plant cells. The specific objective of the proposed research is to elucidate the molecular properties and cellular functions of plant immunophilins and to identify the signaling components from plants that are targeted by immunophilin-ligand complexes. Towards these goals, cDNAs for plant immunophilins will be isolated and characterized. Detailed analyses of spatial and temporal patterns of immunophilin expression will be performed to provide clues on the functional significance of these proteins during plant development. Three approaches will be exploited to address the function of immunophilins in plant cell and development. One is to search for the partner proteins for immunophilins using affinity chromatography and yeast two-hybrid system. The second approach is to generate transgenic plants that are altered in the expression level of immunophilins and to monitor the phenotypic, cellular, and molecular changes of the plants (so called reverse genetic analysis). The last approach will utilize cell free systems to test whether plant immunophilins are involved in protein folding and trafficking as some of animal and yeast homologues. As a key step to dissect the immunosuppressants-sensitive signaling pathways in plant cells, the functional targets for these drugs will be identified and characterized at the molecular level . These studies will not only reveal the multifaceted nature of plant immunophilins but should also provide insight into immunophilin functions in mammalian cells.

A Novel Ca2+ Sensor and Its Target Kinase in Arabidopsis

A number of stressful factors such as drought, cold, salinity, can severely hamper plant growth and development. To survive these extreme conditions, plants have evolved complex mechanisms to "monitor" the stress conditions and respond by changing their physiological and developmental programs. The long term goal of this project is to understand the signaling pathways that link the environmental stress factors to cellular responses in higher plants.
Almost all extracellular signals, including plant hormones, light, stress factors, and pathogenic or symbiotic elicitors, can elicit Ca2+ pulses or transients which serve as "second messengers" in further signaling processes. Because different signals often induce distinct and specific cellular responses, an interesting question is how cells distinguish the Ca2+ messengers produced by different stimuli and respond accordingly. Studies suggest that signaling components that "sense" and "interpret" the Ca2+ parameters hold the key.
The laboratory has identified a new family of Ca2+ sensor proteins that play a role in plant response to stress signals. One member of the family, AtCBL1, is strongly responsive to stress factors including drought, cold, and wounding. Another member, AtCBL4 or SOS3, is required for salt tolerance. To understand the molecular mechanism for AtCBL1 function, the laboratory has made a key finding that AtCBL1 specifically associates with a family of novel protein kinases that are found only in higher plants. These AtCBL1-interacting protein kinases (CIPKs) are highly related to each other and their kinase domains are most similar to SNF1 subfamily of protein kinases. However, all CIPKs contain a unique C-terminal non-kinase domain that is responsible for interaction with AtCBL1. Further studies with one of the CIPKs, CIPK1, show that AtCBL1 interaction with CIPK1 requires micromolar levels of Ca2+, suggesting that Ca2+-binding changes the conformation of AtCBL1 and triggers association with CIPK1. Kinase assays using recombinant CIPK1 determined that CIPK1 is a serine/threonine kinase but has unique cofactor preference. For instance, CIPK1 highly prefers Mn2+ for its activity and functions as a Mn2+-binding protein.
This project will further explore the functional significance of AtCBL1-CIPK1 complex using a combination of biochemical, cell biology, and molecular genetic approaches. In the first objective, biochemical and cell biology approaches will be used to dissect the native form of CIPK1 holoenzyme and identify the functional substrates for CIPK1. In the second objective, molecular genetic approaches using transgenic plant and "knockout" mutant models will be used to unravel the function of AtCBL1-CIPK1 in plant growth and developmental processes especially under stress conditions. This study will establish a new paradigm for Ca2+ signal transduction, which will have major impact on cell biology in general and on signal transduction research in particular.

Reversible protein phosphorylation is the most common mechanism for cellular regulation in eukaryotic systems. Studies have demonstrated that serine/threonine phosphorylation plays a key role in the regulation of plant physiology and development. However, tyrosine phosphorylation, despite its overwhelming importance in animals, was largely neglected in higher plants. Recently the first protein tyrosine phosphatase (PTP) has been characterized from higher plants. Furthermore, a diverse group of 21 genes encoding putative PTPs have been identified from the Arabidopsis genome. Genetic analyses of 4 PTPs have demonstrated that they serve critical functions in plant development, hormone signaling, and stress responses. These studies have broken new ground in the understanding of tyrosine phosphorylation in plants and opened up a new area of signal transduction research. In this project, a multifaceted approach is taken that combines most advanced technology in proteomics and genomics with existing procedures in biochemistry, molecular and cellular biology, and genetics to address the function of tyrosine phosphatases in Arabidopsis. The cDNAs for all PTPs will isolated, enzymatic properties of these putative PTPs will be characterized, their expression pattern and subcellular localization will be determined, and genetic models will be produced and characterized. To understand the molecular mechanism underlying PTP function, the in vivo substrates of PTPs will be identified using mass spectrometry. A useful database on phosphoproteome of Arabidopsis will be built and will be publicly accessible through PlantP website (http://plantsp.sdsc.edu/).
The goal of this project is to understand the function and interplay of all tyrosine phosphatases in plant growth and developmental processes at both whole plant and molecular levels. Such information will contribute to the overall goal of 2010 program: to understand the function of all Arabidopsis genes. This project will also integrate educational activities into the research program by establishing a summer workshop for high school biology teachers.

Plants cannot move and therefore they need to deal with environmental changes on-site. All plants that survive to this day have learned many ways to adapt to environmental changes, especially stressful conditions that affect their growth and development. If one understands how plants adapt to environmental stress, such knowledge can be used to engineer plants with strongest tolerance to the stress conditions--supercrops--to benefit agriculture. This project aims to find out the molecular mechanisms underlying plant responses to environmental stress factors. Almost all extracellular signals, including plant hormones, light, stress factors, and pathogenic or symbiotic elicitors, can elicit Calcium (Ca2+) pulses or transients which serve as "second messengers" in further signaling processes. Because different signals often induce distinct and specific cellular responses, an interesting question is how cells distinguish the Ca2+ messengers produced by different stimuli and respond accordingly. Studies suggest that signaling components that "sense" and "interpret" the Ca2+ parameters hold the key. Recent studies identified a new family of Ca2+ sensor proteins (CBLs) that play a role in plant response to stress signals. This family of calcium sensors works by interacting with a family of protein kinases (CIPKs). This project will use a number of approaches including biochemical, cell biological, genetics, and genomics tools, to understand how this CBL-CIPK network transduces the signals from the outside to the inside of plant cells.
This research will involve undergraduate students who will learn first-hand biological research skills in the lab. In addition, this project will be educating high school biology teachers who will participate in lab experimental work during the summer. Such outreach activity will provide a link between K-12 schools and university research labs and will bring current research topics to the K-12 classrooms.

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Luan
This award supports the PI and two postdoctoral students from the University of California, Berkeley in a collaboration with Jorg Kudla of the Department of Molecular Botany at the University of Ulm, Germany. The research will focus on understanding the signaling pathways that link environmental stress factors to cellular responses in higher plants. These responses are an evolutionary development that allow plants to "monitor" potentially harmful stress conditions and then change their physiological and developmental programs accordingly. The PI's recent studies have identified a new family of doubly ionized Calcium sensor proteins that play a role in plant response to stress signals. One member of that family is probably instrumental in the plant response under study.

The combined effort of the US and German groups is indispensable to raising the level of research to a new height. The collaboration will also allow for international training of the two US postdoctoral students involved in the research.

Scientific Merit
Starch is the ultimate storage molecule formed in the photosynthetic fixation of carbon dioxide by chloroplasts. Starch accumulates during the day and is degraded at night to intermediates that are exported to heterotrophic organs. Although the biosynthesis and degradation of glycogen in animals are regulated by protein phosphorylation via mechanisms that are textbook material, the mechanism by which diurnal cycles control the biosynthesis and degradation of transitory chloroplast starch has long remained a mystery. In a recent study, this group obtained evidence that a dual specificity protein phosphatase, DSP4, binds to starch granules during the day and dissociates at night. Disruption of the DSP4 gene resulted in a dramatic increase in the level of starch in mutant Arabidopsis plants. Two regulatory factors linked to light and dark i.e., pH and
redox status changed both the activity and the starch-binding capacity of DSP4. The results further revealed that DSP4 represents a major fraction of granule-bound phosphatase activity during the day but not at night. The strategy used suggests that DSP4 acts as a bridge between light-induced redox changes and protein phosphorylation in the regulation of starch accumulation.

This is also the first study that establishes a functional connection between protein phosphorylation and starch metabolism, providing a stepping stone for further understanding the mechanism underlying starch balance in plants. This project will: (1) identify the mechanism of DSP4 regulation by the light-mediated redox system, (2) characterize the potential target GSK3-like kinase and its function in starch metabolism, (3) explore the proteome on the starch granule and determine which proteins are modified by protein phosphorylation and regulated by DSP4, (4) determine whether other DSP4-related protein phosphatases function in starch metabolism. Information gained in these studies will be fundamental to understanding the molecular mechanism underlying diurnal regulation of starch metabolism in the chloroplast.

Broader Impact
In addition to contributing to the body of fundamental science, this research will impact society as well as education. Undergraduate students associated with the project will apply findings made in their research to food improvement via an ongoing multi-institutional project in the Co-PI's laboratory. This research is designed to improve the nutritional properties of sorghum a grain that serves as a major food staple for the world's poorest people. If the findings made with Arabidopsis can be extended to sorghum, the work of undergraduates affiliated with this NSF research could have significant impact on humanity and world agriculture. The research will have an additional effect on undergraduate education through major courses the PIs teach at Berkeley and through independent research programs in PIs' laboratories. The NSF project will also significantly enhance graduate education not only through direct lab training but also through the existing international collaboration in plant biology between the Unversity of California, Berkeley and the Chinese Academy of Sciences, which will positively influence the experience of graduate students at the international level. Finally, the project will assist ongoing efforts focused on local high schools to encourage minority students to obtain a higher education and enter biology.

2010 Arabidopsis/AFGN Collaborative Project: "An exemplary calcium signaling network in plant abiotic stress responses"

US-PI: Sheng Luan, University of California at Berkeley, USA
German-PI: Jorg Kudla, Universitat Munster, Germany

The calcineurin B-like calcium sensors (CBLs) and their interacting protein kinases (CIPKs), collectively named CBL-CIPK network. URL where the public can get information on the project:
http://www.uni-muenster.de/Biologie.Botanik/agkudla/kudla.html
Definition of Determining the Function: Recent studies by the investigators uncovered a new family of Ca2+ sensors (CBLs) that target a family of protein kinases (CIPKs), establishing a novel paradigm for Ca2+ signaling in plants. The Luan-Kudla collaborative research supported by NSF and DFG has established an extensive CBL-CIPK network that functions in a number of signaling pathways including responses to abiotic stress, nutrient sensing, and ABA responses. This research seeks to perform a systems analysis of the mechanisms and functions of CBL-CIPK interaction network and its contribution to the integration and decoding of various calcium signals during abiotic stress responses.

The functional analysis results will be shared through the webpage above and publications as the results come along. Significance: Almost all signal transduction processes in plants involve Ca2+ that serves as a vital second messenger. Understanding how Ca2+ mediates the cellular responses triggered by myriad environmental signals is one of the most important goals for plant biologists in years to come. Besides shedding light on the molecular basis for plant tolerance to environmental stress factors, this research will produce insights into calcium signaling mechanisms with relevance for all eukaryotes, provide novel tools for protein-protein interaction, mutant analyses, and cell biology research in plant biology.

Broader Impact: In addition to contributing to the body of fundamental science, this research will impact society as well as education. The findings made in this research can be applied to crop improvement via ongoing multi-national collaborations in the Luan laboratory. This research is designed to improve the nutrient uptake and stress tolerance of crop plants especially rice that serves as a model for cereals. The research will have an additional effect on undergraduate and graduate education through major courses the PI teaches at Berkeley and through independent research programs in PI's laboratory. The NSF project will also enhance ongoing efforts focused on local high schools to encourage minority students to obtain a higher education and enter biology. Furthermore, the US-Germany collaboration will provide a critical platform for training students and postdocs in international collaboration, a crucial component of education in today's global village atmosphere.

PI: Sheng Luan (University of California, Berkeley)

CoPI: Peggy Lemaux (University of California, Berkeley)

Key Collaborators: Rod Wing (University of Arizona)and Bin Han (National Center for Gene Research, Chinese Academy of Sciences)

Plant growth minimally requires sunlight, CO2, water, and minerals. While sunlight and CO2 are abundant, water and minerals are major limiting factors for crop production. Although extensive research effort has been directed to studying water use efficiency and drought tolerance, much less effort has been expended on understanding the mechanisms of plant nutrient use efficiency and tolerance to low-nutrient status in the soil. Not knowing how to increase nutrient use efficiency in crops resorts to heavy use of fertilizers, a practice that endangers environment and sustainable agriculture. The overall goal of this project is to provide a comprehensive understanding of the gene networks responsible for high potassium (K)-use efficiency in cereal crops using genome-wide approaches. Rice is chosen as a model because of its rich genetic and genomic resources and its ease of transformation. The specific objectives are as follows: (1) to characterize the biological roles of all K-transporters encoded in the rice genome. As K+ is not metabolized, effective uptake and distribution of K+ hold the key for high K+ use efficiency in plants. Dissecting the roles of all K-transporters in rice will generate a "road map" of K+ movement throughout a cereal crop and provide possible ways to improve efficiency; (2) to identify genetic loci and individual genes associated with low-K tolerance in rice. Rice landraces display dramatic natural variations in low-K tolerance and such variations can be linked to genes using sequencing-based association mapping that would speed up the identifications of QTLs; (3) to conduct RNA-seq-based comparative transcriptome analysis and identify genes involved in low-K response and adaptation. By comparing transcriptomes of "highly tolerant" and "highly sensitive" landraces to the low-K condition, differentially expressed genes between the two phenotypic groups will be identified and their relevance to phenotypic variations will be scored; and (4) to integrate the datasets from Objectives 1-3 to assemble a gene regulatory network. This transformative knowledge will lay the foundation for marker-assisted classical breeding as well as genetic engineering approaches to improve K-use efficiency in cereal crops and reduce the use of fertilizers in crop production.

A number of broader impacts to society and education will be achieved. Rice is one of the most important food crops, feeding more than half of the world's population. Understanding the mechanisms of low-K tolerance in rice will provide a knowledge base for breeding rice varieties (and other cereals) that produce high yield with minimal fertilizer use, protecting environment and contributing to sustainability of agriculture. This project integrates undergraduate and graduate education into all aspects of the research. Outreach activities will be undertaken that include training of underserved high school and community college students through collaboration with Biotech Partners and STEM program to introduce these students to modern biology techniques and knowledge with the goal of increasing their interest in science careers. All sequence data and annotations will be accessible through the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/). All sequences generated from expression profiling will be deposited at GenBank, the Plant Expression Database (http://www.plexdb.org/) and PlantGDB (http://www.plantgdb.org/).

To grow, plants require a balanced supply of mineral nutrients, but today's soil is often deficient in essential minerals such as nitrogen, phosphorus, and potassium. To overcome this deficiency, crop production in the U.S. relies heavily on use of fertilizers, but this practice is costly, endangers environmental health and is not sustainable, because some essential minerals (e.g., potash and phosphorus rock) are limited natural resources. An alternative and more sustainable agricultural strategy is to breed plants with new traits that allow them to grow in nutrient-imbalanced soils. To achieve this goal requires better understanding of the molecular networks that allow plants to respond and adapt to the constantly changing status of mineral nutrients in the soil. This project addresses this need by studying how plants maintain the proper balance of mineral nutrients when the soil contains too much or too little of specific minerals, thus establishing the knowledgebase for breeding crops with little need of fertilizers, thereby supporting sustainable agriculture. The project will have educational benefits by providing opportunities for undergraduate and graduate students to engage in research and by reaching out to high school students, including those from underrepresented groups, through workshops and other activities aimed at promoting interest in biology research.

Plants constantly monitor nutrient status in the soil and maintain nutrient homeostasis by controlling ion transport across the plasma membrane (for uptake) and tonoplast (for storage). However, the signaling mechanism linking nutrient status in the soil and membrane transport in plants is largely unknown. The principal investigator's laboratory discovered the CBL-CIPK calcium signaling network that functions in a number of cellular pathways including nutrient sensing. In addition to the CBL-CIPK pathway that targets a voltage-gated potassium channel in response to low-potassium status, their recent studies further identified a complex CBL-CIPK network that regulates vacuolar transport of a number of mineral nutrients. The findings have thus connected the CBL-CIPK signaling mechanism to the regulation of transport activities at the plasma membrane and vacuolar membrane, the two most important sites for nutrient homeostasis in plant cells. The proposed research will test the major hypothesis that a large network of "CBL-CIPK-ion channel" interactions couples the environmental signals, e.g., changes in nutrient status, to the regulation of membrane transport and nutrient homeostasis in plant cells. A combination of approaches, including genetic, biochemical, and electrophysiological tools, will be used to address connections of the CBL-CIPK network to potassium and magnesium homeostasis with a focus on understanding the coordination between the plasma membrane and vacuolar pathways in the regulation of nutrient balance.

Abstract This project aims to support attendance of young faculty, under-represented minority scientists, graduate students and post-docs at the 2019 Gordon Research Conference (GRC) and associated student/postdoc-run Gordon Research Seminar (GRS) on ?Organellar Channels and Transporters.? These GRS and GRC will be held in West Dover, Vermont, on August 3-4 and August 4-9, 2019, respectively. These meetings will bring together scientists from many disciplines working on membrane transport proteins from a wide variety of intracellular organelles in animal, plant, and fungal systems. They aim to capture the most recent discoveries in the field and the emerging trends of integration among different organelles and processes, as well as their relevance for the understanding and treatment of common and rare human diseases associated with the dysfunction of organellar channels and transporters. The meeting will consist of platform sessions, short talks, poster sessions, round tables, communal dining, and plenty social times and conversations that will provide ample opportunity for sharing latest research results as well as technical and conceptual advances, for exploring collaborative possibilities, and for identifying new ways to treat human diseases. We request funds from NCATS (as Primary Institute) and NIAMS (Secondary Assignment) to support junior invited speakers (assistant and associate professors) who have achieved breakthrough discoveries at their early career stage; to facilitate attendance of graduate students, postdoctoral fellows, and junior faculty, including but not limited to women and/or other minorities at the GRS and GRC; and to foster productive scientific and career consulting discussions between senior and young scientists.

Calcium (Ca) is a second messenger in all eukaryotes. Defects in Ca signaling cause numerous human diseases including Alzheimer?s disease, heart failure, metabolic diseases, immune disorders, neurodegenerative diseases, and cancer. Despite the importance and broad medical implications, Ca signaling mechanisms remain unclear. The challenging question concerns how Ca encodes specific information coming from different primary signals and translate them into distinct cellular responses. Coding and decoding the specificity of Ca signals remains a long-standing puzzle in the signal transduction field. The PI?s laboratory studies Ca coding and decoding mechanisms using Arabidopsis as a model system and has made breakthroughs in dissecting Ca- coding mechanisms, setting the stage for this application. The proposed studies seek to understand Ca-coding mechanisms in the contexts of pollen tube growth and innate immunity both of which involve cyclic nucleotide- gated channels (CNGCs) in Arabidopsis. The Specific Aim 1 will address the relationship between CNGC-based Ca oscillations and peptide signaling during pollen tube growth. PI?s lab identified two CNGC-type proteins and calmodulin (CaM) forming a Ca ?oscillator? in pollen tube growth that also requires autocrine peptide hormones produced by pollen tube. The overarching hypothesis is that peptides bind to their receptors that in turn modulate Ca-oscillator channels. This will be tested through genetic analysis combined with single cell Ca imaging. The Specific Aim 2 will identify Ca transporters that work together with CNGCs in immunity signaling. The importance of Ca signaling has long been recognized in innate immunity for both animal and plant cells. PI?s lab identified a CNGC-type channel that generates cytoplasmic Ca spike in response to bacterial pathogens. Using genetic analysis in Arabidopsis and yeast genetic complementation models, Aim 2 will identify the transporters responsible for removing the Ca signal and study how they coordinate with CNGC-type channels to precisely shape the spatial and temporal dynamics of Ca codes. Specific Aim 3 seeks to understand the mechanisms for activation and inactivation of plant CNGCs. The CNGC-type channels function in both pollen tube and immunity models, but they consist of different subunits and their regulations by CaM are different too. Further, while animal CNGCs are activated by the cyclic nucleotides (cAMP/cGMP), the plant CNGCs in pollen tube and immunity models are insensitive to these nucleotides. The hypothesis is that plant CNGCs are regulated differently from animal counterparts and CaM-based regulation depends on subunit composition of the CNGCs. This hypothesis will be tested in Aim 3 using biochemical and electrophysiological approaches in both pollen tube and immunity model. Arabidopsis is an ideal model to address basic Ca signaling mechanisms, as it provides a plethora of genetic tools and an array of whole-organism and single-cell Ca signaling phenotypes in the genetic mutants. Completion of these aims will reveal new Ca coding mechanisms, contributing to the conceptual framework of Ca signaling highly relevant to human health.