Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Plant & Microbial Biology
Non Technical Summary
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 will be deployed to examine the translation landscape of two plant species, tomato, and Arabidopsis, in response to two plant hormones, ethylene, and auxin. A time-course experiment will be performed to maximize the detection sensitivity 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 of two plant species will provide critical information on the conservation of the regulatory elements identified and, thus, inform research on future practical applications.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Plants are sessile organisms that spend their entire lives in one place. As they cannot escape unfavorable environmental conditions, they have developed sophisticated adaptive mechanisms that enable them to endure various stresses by adjusting and coordinating their growth and development with the environment. Understanding these adaptive responses is critical for coping with the agricultural and environmental consequences of climate change, and major research efforts have been directed towards elucidating and exploiting the environment-triggered phenotypic plasticity of plants. The picture that is emerging suggests that a small set of plant hormones function at the heart of the signal integration process. In other words, to convert environmental signals into local or global changes in growth rates and patterns and, hence, into altered developmental outputs, plants manipulate hormone production, distribution and/or response. There are many reports that illustrate how plant hormones mediate phenotypic changes in response to environmental conditions. For example, auxin and ethylene have been shown to play key roles in modulating root architecture under different biotic and abiotic stresses.Based on the central role plant hormones play in mediating responses to the environment, it can be argued that the identification of the genetic hubs where environmental signals are integrated or "plugged" into the hormonal response should be critical for understanding how plants respond to the changing environment9. The characterization of such signal integration hubs will not only improve our basic understanding of this important biological process but also inform the future development of agricultural strategies focused on mitigating the problems associated with climate change. Traditional strategies for identifying key nodes in gene regulatory networks heavily rely on temporal correlation in mRNA levels under different experimental conditions10. Although these types of studies are clearly useful, they are based on the premise that changes in the mRNA levels of, for example, a transcription factor (TF) are followed by changes in the levels of the corresponding protein. However, this assumption is often incorrect, as changes in mRNA levels are not very good predictors of changes at the protein level. Importantly, the development of the ribosome footprinting technology (see below) has made it possible to measure translation rates (ribosome footprints) and, when combined with transcriptomic data (RNA-seq reads), to determine translation efficiency (normalized ribosome footprints/normalized RNA-seq reads) at a genome-wide scale. These quantitative translation parameters provide a better estimate of protein levels. We have recently implemented the ribosome footprinting technology in plants and shown that not only can we measure translation rates and efficiencies of thousands of genes, but also that the gene expression regulation at the translational level plays a critical role in the response to plant hormones (see below). Most importantly, we were able to identify a new node of interaction between ethylene and auxin (see below) that could not have been found by using traditional transcriptional strategies alone. Furthermore, the detailed characterization of one of the translationally controlled genes uncovered a cis-regulatory element with novel regulatory properties, having obvious prospective applications in biotechnology and synthetic biology.Towards the general goal of understanding how diverse arrays of molecular signals are fed into the hormone gene networks, we propose a systems approach that combines temporal transcriptional and translational information at the whole-genome scale.In order to achieve these general goals, we propose the following Specific Aims:AIM 1) Identify genes affected at the translational level in response to ethylene and/or auxin in both Arabidopsis and tomato seedlings.AIM 2) Determine the hierarchical relationships between ethylene and auxin transcriptional and translational responses.AIM 3) Identify cis-regulatory elements involved in translational regulation.Aim 4) Determine the physiological significance of the translation regulation mediated by the identified cis-elements and their potential as signal integration nodes.
Project Methods
AIM 1) Identify genes affected at the translational level in response to ethylene and/or auxin in both Arabidopsis and tomato seedlings. A time course will be used to capture early, transient, and late effects of ethylene and auxin on translation efficiency at the genome-wide scale. To capture the hormone-triggered changes in translation efficiency, the levels of mRNA and the corresponding translation rates will be determined providing the needed tools to evaluate the practicality and efficacy of creating gene network models that integrate transcription and translation effects.AIM 2) Determine the hierarchical relationships between ethylene and auxin transcriptional and translational responses. By comparing the ethylene effect in the presence and absence of the auxin biosynthesis inhibitor kynurenine and the auxin effects in the presence and absence of the ethylene receptor antagonist 1-MCP, hierarchical information will be overlaid with the temporal information created in AIM 1. Again, this information will be used to identify genes, such as EBF2, whose translation efficiency is regulated independently by both ethylene and auxin, and therefore represent potential points of interaction between these two hormones. The transcriptional and translational data generated will also be used to refine the transcriptional and translational gene network models from AIM 1 and to determine the value of examining translation rates in these types of studies.AIM 3) Identify cis-regulatory elements involved in translational regulation. A small set of 5-10 genes will be selected based on the severity of hormonal effects on their translation efficiency and the network analysis from AIMs 1 and 2. The 5'UTRs, CDSs and 3'UTRs of these genes will be individually fused to a reporter gene (GFP) and expressed under the control of a strong constitutive promoter. Changes in the GFP protein levels in response to the different hormonal treatments will be monitored by fluorescence and quantified by Western blot. The levels of total mRNA and polysomal RNA will be relied upon to evaluate the effects at the translation efficiency level. The functional conservation of the identified cis-elements between plant species will be examined by comparing the results of, for example, the constructs carrying the tomato cis-elements expressed in tomato and in Arabidopsis, and vice versa.Aim 4) Determine the physiological significance of the translation regulation mediated by the identified cis-elements and their potential as signal integration nodes. Recombineering technology will be used to tag the candidate signal integration genes identified in AIMs 1 to 3 with a reporter gene, GFP. Next, potential cis-regulatory elements found in AIM 3 to be responsible for this integration function will be replaced by their canonical equivalents (for example, a 3'UTR of a gene of interest will be changed to the NOS terminator) maintaining the rest of the native gene-GFP fusion sequences intact, including potential long-range regulatory sequences located 10-20 Kb away from the coding regions.