Adaptation to the environment is fundamentally important to plants since, unlike animals, they cannot move. Such adaptation often is accompanied by changes in plant phenotypes (appearance) and gene expression, some of which can be controlled by epigenetic mechanisms involving changes in chromatin (DNA or histones) or small RNAs. This research will identify epigenetic changes induced in the model plant Arabidopsis thaliana in response to high ambient temperatures and carbon-dioxide concentrations that lie within the ranges that the Intergovernmental Panel on Climate Changes predicts will occur at the end of this century. This work particularly may identify so-called "global-warming" genes responsible for high temperature and CO2 induced variations in plant phenotypes. High throughput deep sequencing, in combination with bioinformatic analysis, will be used to identify epigenetic changes in cytosine methylation, histone modifications, non-coding small RNAs expression, and mRNA expression. This study will demonstrate the value of this approach for investigating how Arabidopsis adapts to other biotic- and abiotic- environmental factors, such as viruses, fungi, bacteria, drought, cold, and salt concentrations. Further, it might well guide similar studies in crops and biofuel-plants, such as soybean, poplar, and switchgrass.

Broader Impacts. The research will contribute to broadening the education and training of postdoctoral researchers, graduate and undergraduate students by introducing students to the latest concepts and methodologies in epigenetics, genomics, and molecular biology. Furthermore, it will offer them a unique platform for understanding the fundamental impact that global warming might have on plants, and how the genome and environment interact to allow plants to adapt. Presentations about this research will be made to the lay public via the Summer Sundays at Brookhaven National Laboratory Program for scientific outreach.

Project Report

DNA methylation can alter the interactions between proteins and DNA, including transcriptino factors and gene regulatory regions, which affects gene expression. Small RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), can silence genes by cleaving mRNAs and suppress translation. SiRNAs can also mediate RNA-directed de novo DNA methylation. Adding or removing methly,acetyl, ubiquitin, or phosphate groups on histone tails can alter the local chromtin state, which affects access to genetic loci. These three layers of modification are interrelated, proving cells with a dynamic and plastic mechanism for responding to develomental and enironmental cues, which is particularly important for plants given their sessile nature. Elevated CO2 in the atmosphere have enhanced the greenhouse effect and resulted in elevated earth surface temperature and climate change. There is widespread agreement that increases in temperature and atmospheric CO2 can and will have major effects on plant growth and development. However, there are many unknowns, including the direction of the response (positive or negative), how responses change if both temperature and CO2 vary, and the underlying molecular perturbations that lead to the phenotypic changes. In this project, we have shown that elevated temperature or CO2 levels alter the epigenome such as genomic DNA methylation, small RNA expression, and histone modification. These changes may change gene expression, the dynamics of gene regulatory networks, and plant phenotypes. In this study, we investigate these questions using Arabidopsis thaliana, a small flowering plant that only needs about six weeks to go from germination to seed maturation. Using the next generation of sequencing in combination with statistical and computational analyses, the project provided the first genome-wide profiling that demonstrated that doubling the atmospheric CO2 concentration and a 3-6oC increase in temperature from optimum plant growing temperature, the ranges that will occur within this century, can alter the expression of four functional groups of miRNAs controlling flowering time, cell division and proliferation, stress responses, and potentially cell wall carbohydrate synthesis. Particularly, the expression changes were found in all components of the miR156- and miR172-regulated transcriptional factor network in a correlative manner, which could result in elevated CO2-induced early flowering phenotype; this pathway controls flowering time during normal development. We also find that elevated temperature-induced early flowering goes through a separate pathway controlled by a gene called Flower Locus M. The elevated temperature regulates a different set of miRNAs from those affected by low- or freezing temperature; intriguingly, this set of miRNAs can be also affected by elevated CO2 oppositely in terms of expression directions, i. e. increase or decrease in expression. Given that all four groups of miRNAs can potentially affect plant biomass production and are conserved across multiple species, their identifications provide us a starting ground to improve plant yields, particularly those that are economically important, to meet the coming challenges of climate change. In this project, we also shows that elevated CO2 promotes gross genomic DNA methylation. Using whole genome Bisulfite-sequencing method, we identified differentially methylated positions (DMPs) and regions (DMRs) affected by this condition. These changes modify both portein coding genes and transposons. Some of DMRs locate to gene bodies and regulatory regions of the genes that have significant expression changes, therefore might play important roles in plant adaptation to climate change conditions. Since the numbers of genes that show significant expression changes under elevated CO2 or temperature are much larger than those regulated by miRNAs and DNA methylation, other mechanisms such as histone modification and transcription factors might be also involved. Indeed, our studies on two flowering gene loci, FLC and FLM have demonstrated that histone modifications can be altered by these conditions. We also analyzed Arabidopsis phenotypes affected by elevated CO2 and temperature, confirming the opposing effects of these two conditions on Arabidopsis plant biomass and seed productions. Further genetic investigation on identified epigenetic changes will shed new light on how they affect biological pathways in adaption to climate change. Sequencing data analyses were carried out by three Ph.D. and one Master graduate students in the Department of Applied Mathematics and Statistics at Stony Brook University (SBU). Two summer undergraduate interns and three undergraduate students participate plant growing and molecular biology study related to this project. This project has also provided a collaboration opportunity among scientists from SBU, Cold Spring Harbor Laboratory (CSHL), Oak Ridge National Lab (ORNL), University of Texas, and University of Luxembourg in Europe, and resulted in the sharing of facilitates and equipment among SBU, Brookhaven National Lab, CSHL, Yale University, and ORNL. From the scientific standpoint, the strongest broader impact has been on the new level of understanding of how plants respond to environmental stress. The publication resulted from this project, "The Effects of Carbon Dioxide and Temperature on microRNA Expression in Arabidopsis Development," in the journal Nature Communications has opened a new perspective in climate research; its significance was highlighted in multiple news.

National Science Foundation (NSF)
Division of Molecular and Cellular Biosciences (MCB)
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Karen C. Cone
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State University New York Stony Brook
Stony Brook
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