The Role of PKS1 in Root Phototropism A thesis submitted to the Miami University Honors Program and the Department of Botany in partial fulfillment of the requirements for an Honors Thesis and Departmental Honors, respectively By Ashley L. Kuntz May 2007 Oxford, Ohio ii ABSTRACT THE ROLE OF PKS1 IN ROOT PHOTOTROPISM By Ashley L. Kuntz Acquiring information about the surrounding environment is crucial to the survival of all living organisms including plants. Therefore, an incredible diversity of sensory systems has evolved to recognize and relay incoming environmental stimuli such as light, touch, and gravity. Directed growth-based responses in plants to unilateral stimuli are called tropisms. Specifically, light is a major environmental factor that governs the growth and development of plants. To detect and respond to the varying fluence, wavelength, and direction of light, plants have evolved several types of photoreceptors. The phytochromes, in particular PHYA and PHYB in the flowering plant Arabidopsis thaliana, are two main receptors of red light that play important roles in regulating many of the light-induced responses. Photoperception of red light by these phytochromes triggers specific intracellular signaling pathways that induce selective changes in gene expression. Using microarray technology and quantitative Real-Time PCR (qRT-PCR), we found the gene PKS1 to be up-regulated five-fold in response to red light, providing strong evidence that the phototropic response was activated by red light in roots. In order to confirm conclusively that the gene PKS1 was involved in red light phototropism, experiments with the computer-based feedback imaging system ROTATO were performed. Our phototropic studies of roots using the feedback system ROTATO suggested an interaction between PKS1 and PHYA since the roots of the double mutant pks1phyA had negative curvature. The phyA mutant showed an even greater negative curvature. Furthermore, time course experiments using qRT-PCR found that there was a consistent two-fold increase in PKS1 expression to red light exposure. The phyA mutant showed only a 1.2-fold induction level of PKS1 expression with the use of qRT-PCR. Ultimately, we hypothesize that PKS1 mitigates the role that PHYA has in positive and negative root curvature in response to red light. PHYA was identified as the major component involved in regulating PKS1 gene expression in Arabidopsis. iii iv The Role of PKS1 in Root Phototropism By Ashley L. Kuntz Approved by: _________________________, Advisor (Dr. John Z. Kiss) _________________________, Reader (Dr. Quinn Li) _________________________, Reader (Dr. Nancy Smith-Huerta) v vi ACKNOWLEDGEMENTS I want to gratefully acknowledge Dr. John Z. Kiss for his commitment to this project and for his dedication to providing undergraduates, such as myself, the opportunity to pursue research. In addition, I appreciate Dr. Quinn Li and Dr. Nancy Smith-Huerta for their time and comments as readers for my project. Lia Molas has offered helpful advice and taught me many techniques used in this study during my last three years at Miami University. Finally, I thank my family for their constant guidance and support. vii TABLE OF CONTENTS Title Page ii Abstract iii Approval Page v Acknowledgements vii List of Figures and Tables ix Introduction 1 Materials and Methods 7 Results 10 Discussion 16 Literature Cited 19 viii LIST OF FIGURES AND TABLES Figure 1 -- Diagram illustrating the mode of action of the photoreceptor 5 phytochrome Figure 2 -- Diagram of the elements as regulated by red light in Arabidopsis roots 6 Table 1 --Genes displaying robust differences in responsiveness to red light 12 in roots Figure 3 -- Phototropic response to red light of roots in WT and mutants as 13 determined with the feedback system ROTATO Figure 4 -- Time course of PKS1 gene expression to red light in WT using 14 Real-Time PCR Figure 5 -- PKS1 expression in WT, phyA, and phyB mutants after one hour 15 of red light using Real-Time PCR. ix INTRODUCTION Acquiring information about the surrounding environment is crucial to the survival of all living organisms including plants. Therefore, an incredible diversity of sensory systems has evolved to recognize and relay incoming environmental stimuli such as light, touch, and gravity. Directed growth-based responses in plants to unilateral stimuli are called tropisms. In particular, light is a major environmental factor that governs the growth and development of plants. Thus, it is not surprising that plants have evolved a remarkable capacity to track and respond to fluctuations in light quality and quantity (Quail, 2002). Currently, focus has been directed towards a better understanding of phototropism in roots of the flowering plant Arabidopsis thaliana (a member of the mustard family). Phototropism is the directed growth in response to light. For example, it is well known that household plants near a windowsill will grow toward sunlight; thus, these plants have to be rotated on occasion: this is an everyday example of the phototropic response in plants (Whippo and Hangarter, 2006). The growing of an organ toward the source of light is termed “positive” phototropism, while the growing away from the light is known as “negative” phototropism. To detect and respond to the varying fluence, wavelength, and direction of light, plants have evolved several types of photoreceptors. These molecules include the blue/UVA photoreceptors, i.e. the cryptochromes and phototropins, and the red/far-red photoreceptors, i.e. the phytochromes. The phytochromes, designated PHYA to PHYE in 1 Arabidopsis, play important roles in regulating many of the light-induced responses (Molas et al., 2006). Roots typically exhibit a “negative” phototropic response (i.e. grow away from the light) which is induced by blue light. This blue light response is mediated by the phototropins, the same photoreceptors involved in the positive response in stems (Briggs and Christie, 2002). However, Dr. Kiss’s lab recently identified a novel response in roots growing towards a source of red light (i.e. positive phototropism) in Arabidopsis (Kiss et al., 2003). This red-light-based response in roots contrasts to red-light-effects in stems, where red light does not cause a tropistic response but a randomization in growth orientation (Hangarter, 1997). Red light evokes a positive phototropism in roots (Ruppel et al., 2001), and the two main receptors of red light, phytochrome A (PHYA) and B (PHYB), play key roles in this response (Kiss et al., 2003). Photoperception of red light by PHYA and B triggers specific intracellular signaling pathways (Figure 1) that induce selective changes in gene expression thereby driving growth and developmental responses to light signals (Quail, 2002). This red-light-induced response is weak in roots relative to other responses, such as gravitropism and negative phototropism; therefore, it has been difficult to visualize and study. However, a computer-based feedback imaging system can be used to more effectively study this response (Kiss et al., 2003). In addition, a molecular approach to detect genes involved in phototropism can answer questions on the regulation of red- light-induced phototropism in roots. In seedlings and leaves of Arabidopsis, hundreds of red-light-induced genes have been investigated by using microarray analyses, and their 2 regulation by phytochromes has been reported (Kiss et al., 2002; Tepperman et al., 2004; Wang et al., 2002). In an effort to characterize the signaling process of root phototropism, our lab confirmed the presence of red light photoreceptors in Arabidopsis roots. Furthermore, our lab has been studying the gene expression profiling of Arabidopsis roots during the early stages of red light signal. Using microarray technology, there were several red-light-dependent genes of interest identified, whose expression were at least two-fold increased in Arabidopsis roots (Molas et al., 2006). We then evaluated the precise level of expression of four of these genes: NPH3 (Non Phototropic Hypocotyl 3), RPT2 (Root Phototropic 2), PKS1 (Phytochrome Kinase 1) and SPA1 (Suppressor of Phytochrome A Responses 1) using quantitative Real-Time PCR (qRT-PCR). For example, the red-light- dependence of the gene PKS1, by being up-regulated five-fold, provided strong evidence that the phototropic response was activated by red light in roots (Figure 2). PKS1 belongs to a small gene family in Arabidopsis (PKS1-PKS4). It is a phytochrome-binding protein that interacts physically with, and is phosphorylated by, the plant photoreceptor phytochrome. Studies show that light increases PKS1 mRNA levels and concentrates its expression to the elongation zone of the hypocotyl and root. This response is meditated by PHYA acting in the very low fluence response (VLFR) mode. (Lariguet et al., 2003). However, light affects a variety of growth responses in roots including negative and positive phototropism, light-induced gravitropism, root hair formation, lateral root orientation, photo-induction of root growth, and other developmental phenomena (Correll 3 and Kiss, 2002). In order to confirm conclusively that the gene PKS1 was involved in red light phototropism, experiments with the computer-based feedback imaging system ROTATO were performed (Mullen et al., 2000). The ROTATO instrument’s image analysis software is coupled with a motorized vertical rotating stage capable of manipulating a plant seedling to keep any selected region of a root at a prescribed angle indefinitely in spite of curvature generation. Thus, root phototropism can be more effectively studied without the complication of a constantly changing angle relative to gravity. One focus of this research project was to further investigate the induction of PKS1 by red light in roots. From our data, we hypothesize that PKS1 mitigates the role that PHYA has in positive and negative root curvature in response to red light. Furthermore, there was interest to identify which phytochrome played a more significant role in the induction of PKS1 by red light. PHYA was identified as the major component involved in regulating PKS1 gene expression.