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

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

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)

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

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. Ultimately, these studies were performed in order to gain insight into the role of the PKS1 gene in root phototropism.

Figure 1: Summary diagram illustrating the mode of action of the photoreceptor phytochrome. Diagram is taken from Quail, 2002.

Figure 2: Summary diagram of the elements as regulated by red light in Arabidopsis roots. Abbreviations defined include: PKS1 (Phytochrome Kinase 1), SPA1 (Suppressor of Phytochrome A Responses 1), COP1 (Constitutive Photomorphogenic 1), HY5 (Long Hypocotyl 5), ELF4 (Early Flowering 4), and GI (Gigantea).

MATERIALS AND METHODS

Seed Stock

The wild-type Arabidopsis thaliana of the Columbia ecotype was used in these studies. The mutants (of the Columbia background) utilized were phyA, phyB, pks1, and pks1phyA, and they were obtained from Professor Christian Fankhauser’s laboratory at the University of Lausanne, Switzerland (Fankhauser et al., 1999). Seeds used in these experiments were stored at 40C.

Culture Conditions

All Arabidopsis seeds used in these experiments were surface sterilized for 5 min with 70% (v/v) ethanol, 2 rinses with 95% (v/v) ethanol, 8 min with 30% (v/v) commercial bleach and 0.02% (v/v) Triton-100 mixture, and then 4 rinses with water.

These seeds were then sown onto a nitrocellulose membrane surface. All plants were grown in pH 5.5 Arabidopsis growth medium (Kiss et al., 1996).

DNA microarray procedures

Gene profiling via DNA microarrays was performed according to the procedures of Molas et al. (2006).

Phototropic studies with ROTATO

7 In these experiments, WT and mutant lines of Arabidopsis: phyA, pks1, and pks1phyA, were in the Columbia ecotype background. The dish containing one seedling was attached to a vertical stage in the dark, and growth was analyzed with a digital imaging system described by Mullen et al. (1998). A computer-based feedback system was used to constrain the root tip angle to the vertical during unilateral 360 minutes red light stimulation perpendicular to the root axis, as described by Mullen et al.

(2000) and Kiss et al. (2003). Statistical differences in phototropism responses were performed in SAS software and determined by analysis of covariance (ANCOVA).

Real-Time PCR procedures

Total RNA was extracted from the roots, and cDNA was synthesized according to

SuperScript II RNaseH- kit protocol (Invitrogen) on 500 ng of RNA with oligo dT as a primer. Real-Time PCR was performed using Rotor Gene RG 3000 (Corbett Research).

The results were produced by Rotor Gene 6.0 software, and gene expression data was calculated using the Standard Curve method (Livak, 1997).

PKS1 Time Course studies

Seedlings of the plant Arabidopsis (Columbia ecotype) were grown for 4 days in the dark, and then red light treatment was applied. Plants were harvested by excising the roots and placing them into liquid nitrogen. The roots were then stored in –80ºC. RNA isolation and purification were performed using RNeasy plant Minikit (Qiagen). Real- time PCR procedures were performed after RNA isolation and purification.

PKS1 Expression in WT, phyA, and phyB

Seedlings of the plant Arabidopsis (Columbia ecotype) were grown for 4 days in the dark, and then 1 hour of red light treatment was applied. Plants were harvested by pouring RNAlater (Ambion) directly into the plates. Roots were then excised in a laminar flow hood and stored in RNAlater at –80ºC. RNA isolation and purification were performed using RNeasy plant Minikit (Qiagen). Real-time PCR procedures were performed after RNA isolation and purification.

RESULTS

Effects of red light on gene expression in roots.

Through microarray studies, a list of candidate genes that displayed differences

(at least two-fold) in response to one hour of red light in Arabidopsis (Ler ecotype) roots was complied (Table 1). The precise level of expression for four genes: NPH3 (Non

Phototropic Hypocotyl 3), RPT2 (Root Phototropic 2), PKS1 (Phytochrome Kinase 1) and SPA1 (Suppressor of Phytochrome A Responses 1) was then examined using quantitative Real-Time PCR (qRT-PCR). In particular, the gene PKS1 was shown to be up-regulated five-fold. This provided strong evidence that the phototropic response was activated by red light in roots.

Interaction between PKS1 and PHYA.

Previous studies involving Arabidopsis hypocotyl tissue have shown that PHYA is more important to PKS1 regulation than PHYB (Fankhauser et al., 1999; Lariguest et al., 2003; Lariguet et al., 2006). However, experiments have not been performed investigating this regulation in roots (Figure 2). Thus, phototropic studies of roots were completed using the feedback system ROTATO for Columbia wild-type (WT), pks1 mutant, phyA mutant, and pks1phyA double mutant (Figure 3). There was a positive response in the Columbia WT; this angle is smaller than in Ler WT (Kiss et al. 2003). It was shown that the wild-type Arabidopsis of the Columbia ecotype and the pks1 mutant

10 roots had statistically the same root curvature. In comparison, the roots of the double mutant pks1phyA had negative curvature. Interestingly, this suggested an interaction between PKS1 and PHYA. The phyA mutant showed an even greater negative curvature of the roots which was statistically different.

Consistent induction of PKS1.

Time course experiments were performed to evaluate the induction of PKS1 gene expression in Arabidopsis (ecotype Columbia) roots over a period of time when exposed to red light (Figure 4). Seedlings were grown in the dark and then transferred to red light for the indicated times. It was found that there was a consistent two-fold increase in in PKS1 expression from one hour to four hours of red light exposure by using Real-Time

PCR. However, PKS1 expression decreased to a 1.1-fold induction at eight hours.

PHYA regulates induction of PKS1 gene expression.

Because PKS1 is known to be involved in PHYA and PHYB-meditated responses, it was decided to examine the expression levels in roots using Real-Time PCR (Figure 5).

Thus, the induction of the gene was studied in the Columbia WT, phyA, and phyB mutants after the seedlings were grown in the dark and then transferred to one hour of red light (Figure 5). It was discovered that the WT had a 3.8-fold induction of PKS1 expression, and the phyB mutant was up-regulated by 3.1-fold. In comparison to the WT and phyB mutant, the phyA mutant was showed only a 1.2-fold induction level of PKS1

11 expression . Thus, without the presence of PHYA, PKS1 expression was significantly decreased.

Table 1: Genes in known light-regulated pathways displaying robust differences (at least two-fold) in responsiveness to red light in roots as determined by microarray. Response Gene Name AGI Expression directions number* levels

Induced HY5 (Long Hypocotyl 5) At5g11260 14

SPA1 (Suppressor of PhyA At2g46340 6.5 Responses 1) PKS1 (Phytochrome Kinase 1) At2g02950 3.8

RPT2 (Root Phototropic 2) At2g30520 3.3

NPH3 family (Non Phototropic At5g48800 2 Hypocotyl 3)

Repressed NPH3 family (Non Phototropic At1g03010 2 Hypocotyl 3) * Arabidopsis Genome Initiative

12 15

5 ) s e e

r 0 g e d (

-5 e r u t

a -10 v r

u Columbia WT

C -15 pk s1 mutant pk s1phyA mutant -20 phyA mutant

-25 -5 45 95 145 195 245 295 345 395 Time (min)

Figure 3: Phototropic response to red light of roots in WT and mutants as determined with the feedback system ROTATO. Roots of the double mutant pks1phyA had negative curvature, suggesting an interaction between PKS1 and PHYA.

13 3

2.5 n o i s s

e 2 r p x E

f o

1.5 l e v e L

v 1 i t a l e R 0.5

0 1 h RL 2 h RL 4 h RL 8 h RL

Figure 4: Time course of PKS1 gene expression in response red light in WT using Real- Time PCR (n=3). Seedlings were grown in the dark and transferred to red light for indicated times. A consistent two-fold increase in PKS1 expression as found from one hour to four hours of red light exposure.

14 5 4.5

o 4 i s s

e 3.5 r p x 3 E

f o

l 2.5 e v

e 2 L

e v

i 1.5 t a l

e 1 R 0.5 0 WT phyA phyB

Figure 5: PKS1 expression in WT, phyA, and phyB mutants after one hour of red light using Real-Time PCR (n=3). Seedlings were grown in the dark and transferred to red light. PHYA regulates induction of PKS1 gene expression.

15 DISCUSSION

The main goal of this study was to gain a better understanding of the role of PKS1 in root phototropism. PKS1 was investigated using the WT (Columbia), pks1 mutant, phyA mutant, pks1phyA mutant, and phyB mutant.

Interaction between PHYA and PKS1.

By observing the pks1phyA mutant during phototropic studies using the feedback system ROTATO, we propose an interaction between PKS1 and PHYA in root phototropism. The pks1phyA mutant exhibited a negative curvature in comparison to the

WT and pks1 mutant. Phytochromes, such as PHYA, are synthesized in darkness in their physiologically inactive red light absorbing form (Pr). Phytochromes convert from the biologically inactive form (Pr) to active form (Pfr). The light induced conformational change is accompanied by the phosphorylation of proteins, such as phytochrome kinase

1(PKS1); (Frankhauser et al., 1999). Phosphorylation of PKS1 protein is hypothesized to induce a kinase signaling cascade that leads to ultimate action within the cytoplasm and/or nucleus (Smith, 2005).

Furthermore, the phyA mutant showed an even greater negative curvature, which differed from the expected results. PHYA is a predominant phytochrome that senses red light signals in roots; thus, the phyA mutant roots were anticipated to have no response to red light without the presence of this phytochrome. Thus, our hypothesis proposes that

PKS1 mitigates the role that PHYA has in positive and negative root curvature in response to red light.

16 Future research is necessary to confirm the effect of PKS1 on root phototropism, and it would require the over-expression of PKS1. Arabidopsis with the over-expression of PKS1 would clearly show the roots’ curvature tendencies when exposed to red light. If

PKS1 decreased positive curvature, the feedback system ROTATO would show less positive curvature than the Columbia WT. In addition, if PKS1 mitigated negative curvature, ROTATO results would show less negative curvature than in the phyA mutant.

Regulation of PKS1 expression.

In this study, a consistent two-fold up-regulation of PKS1 was found in

Arabidopsis roots that were exposed to red light over a period of time. Our experiments examined PKS1, a phytochrome binding protein that interacts with the phytochrome photoreceptors, at the mRNA level in roots. mRNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. Thus, these results suggest that PKS1 gene expression may be regulated at the mRNA level. Future studies could examine the regulation of PKS1 at the protein level to determine if there are additional points of regulation.

Confirmation of PHYA regulation of PKS1 expression.

Previous studies performed in the Kiss lab established that PHYA and PHYB play a major role in red light induced phototropic response (Kiss et al., 2003) and that PKS1 is highly induced in roots of seedlings after one hour of red light (Molas et al., 2006).

Furthermore, recent photobiological studies showed that PHYA is the predominant

17 phytochrome regulating PKS1 in Arabidopsis hypocotyls (Fankhauser et al., 1999;

Lariguest et al., 2003; Lariguet et al., 2006). Hence, we investigated the participation of

PHYA and PKS1 in red light induced phototropic response in roots. We show that

PHYA and PKS1 are required for a typical response to red light in roots. Interestingly,

PHYA, more than PHYB, plays a predominant role in regulating PKS1 gene expression.

Conclusions.

The major findings of these studies focus on the induction of PKS1 by red light in roots. A hypothesis was established and proposed that PKS1 mitigates the role that

PHYA has in positive and negative root curvature when exposed to red light. In addition, this research examined which phytochrome, PHYA or PHYB, played a more significant role in the induction of PKS1 by red light. Thus, PHYA was identified as the major component involved in regulating PKS1 gene expression. Further understanding of the role of the PKS1 gene in root phototropism was accomplished thorough this research.

However, the precise molecular mechanism of root phototropism is still unknown, and future studies will be developed to address these critical issues in plant biology.

18 LITERATURE CITED

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Hangarter RP. “Gravity, light and plant form.” Plant Cell Environment 20 (1997) 796-800.

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Tepperman JM, ME Hudson, R Khana, T Zhu, SH Chang, X Wang, and PH Quail. “Expression profiling of phyB mutant demonstrates substantial contribution of other phytochromes to red-light-regulated gene expression during seedling de-etiolation.” Plant Journal 38 (2004): 725-739.

Wang H, LG Ma, J Habashi, JM Li, HY Zhao, and XW Deng. “Analysis of far-red light- regulated genome expression profiles of phytochrome A pathway mutants in Arabidopsis.” Plant Journal 32 (2002): 723-733.

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