Date of Award

Spring 2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

Committee Director

Lesley H. Greene

Committee Member

Jing He

Committee Member

Patricia Pleban

Committee Member

Jennifer Poutsma

Abstract

The present and potential future effect of global warming on the ecosystem has brought climate change to the forefront of scientific inquiry and discussion. For our investigation, we selected two organisms, one from cyanobacteria and one from a cereal plant to determine how climate change may impact these biological systems. The study involved understanding the physiological and adaptive responses at both the genetic and protein function levels to counteract environmental stresses. An increase in atmospheric carbon dioxide is a key factor in global climate change and can lead to alterations in ocean chemistry. Cyanobacteria are important, ancient and ubiquitous organisms that can aid in the study of the biological response to increasing carbon dioxide. Climate predictions estimate that by the year 2100 atmospheric carbon dioxide will exceed 700 ppm. In our first study, we looked at the transcriptional effect of high pCO2 on the cyanobacteria, Trichodesmium erythraeum. Total RNA sequencing was used to quantify changes in gene expression in T. erythraeum grown under present day and projected pCO2 concentrations for the year 2100. Two bioinformatics methods were used to analyze the transcriptional data. The results from this study indicate that a substantial number of genes are affected by high pCO2. However, increased pCO2 does not completely alter any one specific metabolic pathway.

As the climate shifts throughout the world, it becomes essential for crops to withstand weather changes. In our second study, we investigated the function of the temperature induced lipocalin (Tatil) from Triticum aestivum, which is proposed to help plants survive adverse conditions. This protein is part of a functionally diverse and divergent superfamily of proteins called the lipocalins; they share a common three-dimensional structure, which consists of an antiparallel β-barrel and a C-terminal α-helix. Lipocalins are found in various organisms with a wide range of functions such as pheromone activity, lipid transport and coloration. Recently, proteins from wheat and Arabidopsis were identified as lipocalins through the elucidation of three structurally conserved regions. The study is particularly timely, as recent studies within the scientific community have shown that at higher temperatures wheat yields will decrease and production will decline by 6% for each 1°C increase. We analyzed the nature of conservation in a large group of sequentially divergent and functionally diverse lipocalins and identified seventeen highly conserved positions as well as built models of the native three-dimensional state of the wheat lipocalin. Based on these computational studies, the wild-type protein and three variants were chosen for a cellular localization study involving site-directed mutagenesis, a gene gun and a confocal microscope. The results provide support for the hypothesis that the L5 loop is involved in the association of the protein with the plasma membrane. We also developed an expression and purification system to produce the wild-type wheat lipocalin protein. Gel filtration chromatography eluted two different sized proteins. Based on the elution volume, one is believed to be the wheat lipocalin trimer while the other one is the monomer. Circular dichroism and fluorescence spectroscopy show that the biological characteristics of the two proteins are different. In the study, Tatil maintains its structure up to approximately 50°C (122°F). In summary, we provide experimental data to better understand mechanistically how microorganisms and plants adapt to environmental change. In cyanobacteria, we show that T. erythraeum adapts to pCO2 increases by up- or down-regulating its genes. In plants, we provide insight into the way in which Tatil interacts with the plant cell membrane as part of its putative function to facilitate robustness in response to temperature increases. The study of Tatil is vital as this protein is believed to help plants tolerate oxidative stress and extreme conditions which broadens our understanding of plant sustainability in different environments.

DOI

10.25777/xktn-6654

ISBN

9781321833287

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