Joseph John Kieber - US grants

Two-component systems are the primary means by which bacteria sense and respond to environmental stimuli. These systems are comprised of a number of distinct elements, namely histidine kinases, response regulators and in the case of phosphorelays, histidine phosphotransfer proteins (HPts). Genes encoding similar proteins to each of these elements have been identified in Arabidopsis, and for the majority of the 35 such genes no function has yet been definitively ascribed. An integrated approach to elucidate the function of these proteins in Arabidopsis is proposed. A combination of gene knockouts and inducible overexpression will be used to assess the roles of these genes in plant growth and development. The mutant plant lines will be characterized in terms of their response to biotic and abiotic factors such as hormones, light, and osmotic stress, and for their pattern of gene expression. Where in the plants these genes are expressed will be determined using a combination of GUS fusions and in situ RNA analysis. The location of the cognate proteins within the cell will also be delineated. To facilitate this localization, a series of 10 monoclonal antibodies will be generated to marker proteins, each of which resides on a distinct membrane. Protein complexes from Arabidopsis will be purified and analyzed to determine the interactions among these elements and to identify novel interacting proteins. Together, these studies will illuminate the signaling pathways in which each of these Arabidopsis two-component signaling elements function and how they interact to control plant growth and development.
The data from these studies will be deposited on a publicly accessible web page that is currently under construction at UNC (http://www.bio.unc.edu/research/two-component/). A link to this web site will be established on the TAIR database, and we will coordinate with TAIR to deposit data as appropriate. The knockout seeds will be made publicly available through deposition in the ABRC Stock Center at Ohio State. The monoclonals raised against the membrane marker proteins will be available for the cost of shipping through UNH and the cell lines will also be deposited with the American Type Culture Collection (ATCC).
In keeping with the goals of the 2010 project, functional analysis of the two-component signaling elements will aid in our understanding of plants at the organismal, cellular, and evolutionary levels. The research will provide functional information on the role of Arabidopsis two-component signaling systems in plant growth and development. The research will serve to define the subcellular location of the two-component signal transduction pathways, the interactions between the two-component signaling elements, and the downstream targets of the pathways. The research should clarify how a signal transduction mechanism that arose in bacteria has been adapted to plant signal transduction.
These studies should uncover the functions of several gene families in Arabidopsis. The proteins encoded by these gene families are predicted to interact and thus our studies should aid in the development of a paradigm for signaling specificity among interacting members of large gene families. In addition, tools will be developed that will be generally applicable in defining the subcellular location of proteins in Arabidopsis.

DESCRIPTION (provided by applicant): Cytokinins are a group of plant hormones that are powerful mitogens and which play a role in many plant developmental processes. An understanding of the molecular mechanisms by which cytokinins act could help to better understand the regulation of cell proliferation in human cells. Almost nothing definitive is known with regard to cytokinin signaling in higher plants. We propose to utilize an integrative approach to this problem, combining molecular, genetic and biochemical approaches to elucidate the elements involved in cytokinin action. The main thrusts of this proposal derive from our isolation of two cytokinin primary response genes, IBC6 and IBC7, which belong to a larger Arabidopsis gene family that is similar to bacterial signaling elements. We will exploit the cytokinin-specific induction of GFP fluorescence in an IBC6 promoter-GFP transgenic line to identify cytokinin-action mutants by identifying seedlings that inappropriately exhibit GFP expression. Preliminary screens indicate that this approach will be successful and that these mutants should help to dissect the cytokinin response pathway. The rapid induction of IBC6 and IBC7, coupled with their similarity to bacterial signaling elements, suggests that these genes may be involved in cytokinin signaling. We will test this hypothesis and other possible functions of this gene family by determining the effect of altered ARR gene expression, both gene disruption and regulated overexpression, on plant development and cytokinin responsiveness. We will also identify and characterize proteins that interact with IBC6 as a means of identifying potential downstream targets and upstream activators. We propose to identify the cis-acting DNA sequences responsible for the cytokinin-response of IBC6 as a means of starting to unravel the mechanisms by which cytokinins regulate gene expression. Once identified, we will use this element to identify additional potential cytokinin response genes and to identify DNA binding proteins responsible for the function of this element. Finally, we will use Affymetrix gene chips to identify novel cytokinin-regulated genes in Arabidopsis.

How cell regulate their expansion is central question in modern biology. In plants, asymmetric cell expansion is a primary determinant of the size and shape of organs. However, very little is known regarding the molecular mechanisms underlying the regulation of cell expansion in plants. A pair of receptor-like kinases from Arabidopsis, called AIK1 and AIK2, has been identified that directly interact with ACC synthase, the enzyme that catalyzes the rate-limiting step in the biosynthesis of the plant hormone ethylene. Genetic studies have revealed that these AIK genes play an important role in regulating cell expansion in root cells. Ethylene promotes radial expansion of plant cells, and the radial swelling phenotype in the aik1/aik2 double mutant was found to be dependent on ethylene. This, coupled with the observation that these proteins directly interact with ACC synthase, suggests that these AIK genes are involved in the interaction between ethylene and directed cell expansion. This interaction will be further explored by precisely delineating the effects of the AIK mutations on the growth characteristics of the root and by determining if these genes directly regulate features of the cell involved in directing cell elongation, such as the cell wall. The role of ethylene in the aik mutant phenotype will be explored using genetic and biochemical approaches. Finally, additional targets of these AIK kinases will be sought using a simple interaction screen in yeast. This research will shed light on how plants regulate cell elongation, which is crucial to understanding the development of plant form.The proposal will also provide interdisciplinary training opportunities for undergraduate and graduate students in diverse areas including cell biology, plant physiology, molecular genetics, and biochemistry.

The gene network: This project focuses on a gene network that regulates plant responses to
cytokinin, a key hormonal regulator of plant growth and development. The initial steps in
cytokinin signaling are mediated by a 'two-component' system that transmits information from
membrane to nucleus. The two-component system makes use of histidine kinases that act as
cytokinin receptors, histidine-containing phosphotransfer proteins that transduce the initial
signal, and response regulators that regulate the signal output. The transcriptional targets of this
pathway as well as other outputs have yet to be determined. This project will delineate the
network of genes and proteins regulated by two-component signaling elements of Arabidopsis.
This two-component signaling network includes the transcription factors that control gene
expression as well as interacting proteins that regulate other aspects of signal output.
Project URL: http://www.bio.unc.edu/research/two-component/default.htm
Functional characterization: Transcription factors will be characterized in terms of the genes that
they regulate. Proteins will be characterized in terms of their protein-protein interactions within
the signaling network. The physiological function of the network elements will be defined in
terms of their role in regulating cytokinin responses as well as seed size, meristem function,
vascular development, leaf and cotyledon morphology, metal ion homeostasis, and circadian
rhythms.
Sharing of results: Genomic information and tools generated will be made available to the
research community through the web and through standard methods of publication in a timely
fashion. Datasets of general interest, that are not made available through publication, will be
deposited on the project web site within two years of their generation.
Significance of work: These studies will define the circuitry by which the two-component gene
network acts to alter plant growth and development. In addition, these studies will reveal
connections between this pathway and other cellular networks. Determining how this integration
is achieved will contribute to an understanding of signaling networks in plants and will serve as a
model for examining other interactions within the plant cell. The techniques and tools developed
during the course of these studies will assist gene studies in Arabidopsis and in other plant
species.
Broader impact: Results from the project will benefit society through the understanding of a
critical gene network that regulates multiple traits of agronomic importance. Elucidation of these
roles will provide avenues to modify such agriculturally relevant traits such as senescence, plant
transformation, grain yield and filling, and patterns of growth and development. The plant
research community will benefit by the optimization of novel research techniques and the
development of new community resources. The proposed research will enhance the
infrastructure of research and education by providing hands-on training for undergraduate
students, graduate students, and post-doctoral researchers within the PIs' labs. In addition, the
PIs will partner will local groups to assist in the creation and maintenance of programs aimed at
fostering science education in grades K-12.

The gaseous hormone ethylene has profound effects on plant growth and development. The rate-limiting and key regulatory step in the biosynthesis of this hormone is catalyzed by ACC synthase (ACS). Previous studies have shown that the C-terminal domain of ACS targets this protein for rapid degradation, and several factors involved in regulating this degradation have been identified. The primary goal of this research is to further elucidate the mechanisms regulating ACS protein turnover. To these ends, the following lines of investigation will be pursued: 1) The role of protein phosphorylation in controlling ACS protein stability will be examined, and the precise portion of the ACS protein required for its rapid degradation will be delineated; 2) The stability of ACS protein during various developmental events that are associated with a rise in ethylene biosynthesis or in response to exogenous cues will be examined to determine if altered ACS protein stability plays a role in controlling ethylene biosynthesis under these conditions; 3) The role that two Arabidopsis DnaJ (a.k.a. HSP40) orthologs play in regulating ACS turnover will be studied; 4) The CIN2 gene, which was previously identified as a gene that is involved in controlling ethylene biosynthesis, will be cloned and characterized; 5) A genetic screen will be performed to identify novel elements involved in regulating ACS protein turnover. These studies will shed light on the mechanism regulating the stability of ACS proteins and how this contributes to the control of the biosynthesis of ethylene. This research will also increase our understanding of the role of DnaJ in higher eukaryotic organisms, and on the mechanism by which this ubiquitous protein acts.

Results from these studies will lead to a deeper understanding of the regulation of ethylene biosynthesis, which may lead to the ability to manipulate the production of this hormone in an agricultural setting and hence improve the quality and longevity of various agricultural products. Furthermore, the regulation of protein turnover has emerged as a central mechanism underlying a variety of biological systems. This research will shed light on this fundamental process and enhance the infrastructure of research and education by providing hands-on training for undergraduate students, graduate students, and post-doctoral researchers. In addition, it will also allow collaboration with the DESTINY Traveling Science Learning Program to assist in the creation and maintenance of a plant based learning module aimed at fostering science education in grades K-12.

This project will provide support for the 18th International Conference on Arabidopsis Research to be held in Beijing, China on June 20 through 23, 2007. Arabidopsis thaliana has been the subject of genetic analysis for more than forty years. Thousands of researchers worldwide study this highly tractable reference plant, which is used to study nearly all aspects of plant biology. The annual International Conference on Arabidopsis Research (ICAR) brings together approximately 1,000 participants from this tightly-linked international scientific community to exchange scientific results and report on progress in the field. This year the 18th ICAR will be held in the Jiuhua Spa & Resort (http://www.jiuhua.com.cn/enindex.asp) in Beijing, China. This will be the first time ICAR will be held in an Asian country, and thus provides unique opportunities for building ties among researchers in Asian countries and the Western countries. The conference will include sessions on cell biology, cell signaling, development, metabolism, proteomics, biotic and abiotic interactions, genetic and epigenetic mechanisms, evolutionary biology, novel tools, and techniques and resources. An important component of the conference is the presentation of current research by approximately 50 speakers and more than 600 posters. The majority of the oral presentations will be by graduate students, postdoctoral researchers and junior faculty. This will promote the training of students and postdocs and provide important opportunities for career development for the selected speakers.

The broader impact of the conference lies in its ability to enhance the exchange of information at the forefront of Arabidopsis research world-wide, creating new networks and collaborations. Conference abstracts will be publicly available on the conference Web site, which is linked to the Arabidopsis Information Resource (TAIR) Web site. In addition, the Conference fosters the participation of minority scientists by providing them with travel subsidies. Furthermore, this year's conference will emphasize extending Arabidopsis studies to other plant species, stimulating innovative strategies for improvement of agricultural plants. There will be many speakers who research does not focus on Arabidopsis as their primary organism. The broader impact of the conference lies in its ability to enhance the exchange of information at the forefront of Arabidopsis research creating new research networks and collaborations. Conference abstracts will be available on the conference webpage and at TAIR (the Arabidopsis Information Resource) providing for public access to this information. A workshop on the use of Arabidopsis in the classroom is being organized. Travel subsidies for conference participants from developing countries and full coverage for all conference costs for underrepresented minorities and participants from Minority Serving Institutions will be offered.

A grant has been awarded to The University of North Carolina at Chapel Hill (UNC) under the supervision of Dr. Alan M. Jones entitled "Rapid Image Acquisition of Dynamic Arabidopsis Cells and For High-throughput Genetic Screens" for the purpose of acquiring and maintaining a specialized microscope. The enormous progress made in the plant sciences from sequencing the Arabidopsis genome has propelled research to the next higher research plane, where examining how and when cellular proteins interact over time at a high spatial resolution has become the new standard in demand. Both the demand and the need for sophisticated imaging instrumentation have increased, pari passu. However, imaging fluorescently-tagged proteins in plant cells faces several surmountable challenges. One is the dynamic nature of plant cells due to cytoplasmic streaming (rapid cellular movement) that occurs with speeds greater than the speed of standard capture by a standard confocal microscope (which is a specialized microscope that enables one to visualize fluorescently-modified proteins in all kinds of live cells). Therefore, to capture transient protein-protein interactions within plant cells requires a specialized confocal microscope that enables rapid acquisitions nearly simultaneously through the cell, over a short time scale. Reconstitution of this data will be used to produce a 3-dimensional view of plant protein-protein interactions in vivo over time. This grant will enable 8 highly-productive plant scientists at UNC to make significant and faster contributions to fundamental science leading to increased crop production, plant disease resistance, and biofuels production.

The broader impact will occur in the classroom, in public, and in the research labs. The use of a confocal microscope will be introduced to upperclassmen in an intense, hands-on cell biology laboratory. The public will become aware of the power of rapid imaging through an educational display and presentations by the PIs. The plant science group at UNC collectively trains 20 undergraduates, 15 predoctoral, and 30 postdoctoral students each year. The requested instrument will also serve the confocal imaging needs of many additional labs on campus. It is the first of its kind on the UNC campus and only the second like it in the state of North Carolina. Many scientists beyond the plant science group will greatly benefit from this specialized microscope.

Cytokinins are plant hormones that affect a diverse array of plant growth and development processes. How cytokinins mediate such a diverse array of processes remains a fundamental question in plant biology with important agricultural implications. In this project, genetic, molecular, and genomic approaches will be used to identify and characterize the critical factors that control gene expression in response to cytokinin. Computational approaches will then be used to build a network of cytokinin-regulated gene expression. These data will be incorporated into a database available through the project website (http://www.bio.unc.edu/research/two-component/default.htm) so that the research community can easily view and make use of the regulatory network. Results from the proposed research will benefit society through the elucidation of a critical gene network that regulates multiple traits of agronomic importance. Previous work suggests that results from the project will provide avenues to modify agriculturally relevant traits such as grain yield, energy capture and use, senescence, responses to environmental stresses, and patterns of growth and development. The proposed research will enhance the infrastructure of research and education by providing training for undergraduate students, graduate students, and post-doctoral researchers, as well as the creation and maintenance of programs aimed at fostering science education in grades K-12.

Intellectual merit:
The gaseous phytohormone ethylene has profound effects on plant growth and development. The biosynthesis of this hormone is influenced by many other hormonal, developmental, and environmental signals, and as such acts as an important point of crosstalk in regulating plant growth and development. This project will explore the mechanisms regulating ethylene biosynthesis in the model plant Arabidopsis thaliana. Previous studies have shown that the regulation of the stability of ACC synthase, a key enzyme in the production of ethylene, is the critical factor controlling how much ethylene plants make. The research supported by this award will build on these findings by charactering the half-lives of different ACC synthase proteins from Arabidopsis to determine how rapidly each is turned-over and how pairing different isoforms together affects their turnover. How other hormones interact to regulate the stability of these different ACS proteins will also be studied. This will help understand how these various signaling pathways influence the production of ethylene. A final aim is to examine the role of proteins that were previously identified as interacting with ACC synthase, with a focus on how these proteins may further regulate the function of ACC synthase.

Broader impacts:
Results from this research will lead to a deeper understanding of the regulation of ethylene biosynthesis, which may lead to the ability to manipulate the production of this hormone in agricultural settings and hence an improvement in the quality and longevity of various agricultural products. Furthermore, the regulation of protein turnover has emerged as a central mechanism underlying a variety of biological process, and this project will shed light on this fundamental process. These studies will enhance the infrastructure of research and education by providing hands-on training for undergraduate students, graduate students, and post-doctoral researchers. The PI has connections with programs aimed at broadening participation in science. In addition, graduate students are also involved in summer science camp activities for 6-8th graders, providing them hands on science experiments related to plants and ethylene.

PI: Joseph Kieber (University of North Carolina - Chapel Hill)

CoPIs: G. Eric Schaller (Dartmouth College) and Ann Loraine (University of North Carolina - Charlotte)

Cytokinins are plant hormones that regulate many traits of agricultural importance, including the control of grain size and yield. In this project, genomic approaches will be used to uncover how cytokinin regulates growth and development in rice, using this plant species as a model to understand the regulation of grain size and yield in all cereals. Computational approaches will be used to interrogate and integrate the datasets generated by these studies. It is expected that these studies will illuminate the role of cytokinins in panicle development, and will provide specific information on how cytokinins regulate grain development in an agriculturally important crop. Because of conservation of gene sequence among cereals, information obtained from the study of rice may be applied to other agriculturally important species such as maize, wheat, barley, rye, and sorghum.

This project will enhance the infrastructure of research and education by providing training for undergraduate students, graduate students, and post-doctoral researchers. A computation workshop will broaden the ability of the research community to use genomics resources. In addition, through partnerships with local groups including the Montshire Museum of Science (http://montshire.org/), programs aimed at fostering science education in grades K-12 will be created and maintained. Data will be made available through a public database (RiceCytokinin.org) as well as through long-term repositories such as the NCBI Short Read Archive (http://www.ncbi.nlm.nih.gov/sra/) and the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). Germplasm generated in this project will be available upon request and through the USDA Genetic Stocks - Oryza (GSOR) Collection (http://www.ars.usda.gov/Main/site_main.htm?docid=18992&page=1).

Non-technical summary:
Plant cell walls are diverse and highly dynamic structures that respond to developmental and environmental cues. They are a primary source of building materials, paper, biofuels, cotton, and many other useful products and also plays a crucial role in the growth and development of plants. Despite their importance both from a practical and fundamental point of view, the signaling pathways regulating the function and assembly of plant cell walls are just beginning to be understood. This project seeks to gain fundamental knowledge regarding how plants regulate the synthesis of their cell walls, which should ultimately help improve the diverse products derived from them and will provide insight into basic aspects of plant growth and development. In addition to providing interdisciplinary training opportunities for undergraduate and graduate students, this project will work with programs designed to increase the representation of minorities in the sciences and with several outreach programs designed to increase the public understanding of plant science.

Technical Description of the Project:
Previous studies identified a pair of transmembrane protein kinases, called FEI1 and FEI2, that play a role in regulating the biosynthesis of cellulose, which is a critical component of the plant cell wall and acts as the primary load-bearing element. These kinases define a novel signaling pathway that includes a non-canonical role for 1-aminocyclopropane-1-carboxylic acid (ACC), a molecule previously thought to act only as a precursor in the biosynthesis of the phytohormone ethylene. A series of experiments to further characterize the FEI signaling pathway and its role in regulating cell wall function will be pursued. The mechanism by which the FEI pathway regulates cellulose biosynthesis will be explored (Aim 1). The signaling role of ACC and other potential ACC-derivatives in regulating wall synthesis will be examined (Aim 2). Other components involved in this FEI pathway will be identified and characterized, including SHOU4, a protein that appears to act in the FEI pathway and which is likely associated with the cytoskeleton (Aim 3). These studies will answer fundamental questions regarding how this pathway regulates the production of cellulose and how it interacts with other elements to regulate cell wall synthesis. Further, the FEIs are belong to a group of kinases call the receptor-like kinase, which are one of the largest families of genes in plants; the function of only a handful of RLKs are known, and thus these findings will shed light on a poorly understood class of plant signaling elements. Finally, these studies should help elucidate a role for ACC in plant signaling and may uncover important roles for a novel signaling compound in plants.

Plant leaves and flowers are derived from stem cell tissues called meristems. The regulation of meristem development and activity is central to understanding plant growth and development, and impacts many agronomically important traits such as plant architecture, biomass, and grain yield. The goal of this project is to determine how the plant hormone cytokinin regulates meristem function in rice, an important crop and a model system for understanding growth and development of other cereals. For this purpose, the recently developed methodologies for single cell sequencing will be employed. Gene expression in individual cells of the shoot meristem and of the reproductive inflorescence meristem will be examined to uncover cellular mechanisms that regulate the development of leaves, flowers, and grain. In addition, the packaging of the genome will be characterized to identify DNA elements that likely play a role in controlling when, where, and to what degree the genes are expressed in specific cells. Alternations in meristem development, brought about by disrupting cytokinin function will reveal how this hormone orchestrates proper rice development at the cellular level. These studies will illuminate the mechanisms underlying the development of vegetative and inflorescence architectures, with an important emphasis on cytokinin’s role in these processes. In broader context, this work has the potential to realize increased yields in rice and other agriculturally important cereal species. The proposed research will enhance the infrastructure of research and education by providing student training and the development of programs aimed at fostering science education.

The phytohormone cytokinin plays key roles in regulating the activities of the vegetative shoot apical meristem (SAM) and reproductive inflorescence meristem (IM) in rice. Here, the role of cytokinin in regulating the rice SAM and IM will be examined at the single cell and single nucleus levels using an integrative array of genetic, molecular, and single-cell ‘-omic’ approaches. First, transcript abundance and chromatin accessibility in individual SAM/IM cells will be characterized using single-cell transcriptomics coupled with single-nucleus chromatin profiling. This will provide foundational understanding of the regulatory circuits in the SAM and IM. Marker genes and regions of open chromatin within subdomains of the SAM/IM will be correlated to polymorphisms identified in GWAS panels for panicle traits in rice, potentially identifying important genomic regulatory regions for panicle architecture. Second, cytokinin signaling will be genetically perturbed using cytokinin receptor mutants to reveal how this regulatory network influences cell types, their distribution and developmental trajectories, and the activity of genes and regulatory elements within the cell clusters. Third, because type-B Response Regulator (RR) transcription factors control the initial transcriptional output from cytokinin, the direct targets of type-B RRs will be identified by ChIP-seq. Integrating these datasets will identify key targets of cytokinin signaling in specific cells and types of the SAM/IM, a subset of which will then be examined by genetic analysis. Collectively, the proposed studies will result in complementary genome-level datasets that will identify key mediators of cytokinin in regulating meristem activity, potentially revealing emergent properties of meristem regulation.

This award was co-funded by the Plant Genome Research Program in the Division of Integrative Organismal Systems and the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences.

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.