The Pennsylvania State University

The Graduate School

Eberly College of Science

FUNCTIONAL ANALYSIS AND BIOCHEMICAL CHARACTERIZATION OF

HETEROTRIMERIC G PROTEIN IN ARABIDOPSIS

A Thesis in

Biochemistry and Molecular Biology

by

Shiyu Wang

© 2007 Shiyu Wang

Submitted in Partial Fulfillment of the Requirements

for the Degree of Doctor of Philosophy

August 2007

The thesis of Shiyu Wang has been reviewed and approved* by the following: Nina V. Fedoroff Willaman Professor of Life Sciences and Evan Pugh Professor Thesis Adviser Chair of Committee Sarah Assmann Waller Professor of Biology David S. Gilmour Professor of Biochemistry & Molecular Biology

Teh-hui Kao Professor of Biochemistry & Molecular Biology Ming Tien Professor of Biochemistry & Molecular Biology Alan M. Jones George and Alice Welsh Distinguished Professor of Department of Biology University of North Carolina at Chapel Hill, Special Member Richard J. Frisque Department Head of Biochemistry & Molecular Biology *Signatures are on file in the Graduate School.

ABSTRACT

Heterotrimeric G proteins are recognized as important signal transduction

molecules in all eukaryotic organisms from yeasts to humans. Compared to mammals and

invertebrates, simplicity of G protein gene family in Arabidopsis provides a unique

advantage for investigating its functions in various aspects of different physiological

processes. Extensive studies in the last 10 years have shown that the Arabidopsis

heterotrimeric G protein is involved in a transducing a variety of signals, including light,

hormones, biotic and abiotic stressors.

First, we analyzed the functional roles of heterotrimeric G protein in Arabidopsis

oxidative stress responses to ozone (O3). Arabidopsis thaliana plants of homozygous

gpa1-4 (Gα null mutation) are less sensitive to O3 damage than wild-type Columbia-0

plants, whereas agb1-2 (Gβ null mutation) plants are more sensitive to O3 damage than

wild-type Columbia-0 plants. The genes encoding the α and β subunits of the

Arabidopsis heterotrimeric G protein are differentially expressed in the course of the

oxidative stress response to O3, suggesting that they play different roles in the plant’s O3

responses. The first peak of the biphasic oxidative burst elicited by O3 in wild-type plants

is absent in both mutant plants, suggesting that the first peak of the oxidative burst

requires both α and β subunits or the intact heterotrimeric G protein. The second peak is

missing in gpa1-4 mutant plants, but is normal in agb1-2 mutant plants, suggesting that

the second peak of the oxidative burst requires only the α subunit but not the β subunit.

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Thus, the opposing O3 phenotypes and the differential ROS production patterns between

gpa1-4 and agb1-2 mutant plants indicate that the α and β subunits play separable roles

in cell death associated oxidative stress response to O3.

Second, we analyzed the functional role of heterotrimeric G protein of

Arabidopsis unfolded protein responses to Tm. Seedlings of agb1-2 mutant plants with a

null mutation in the gene coding for the β subunit of the heterotrimeric G protein are

more resistant to growth inhibition by the protein glycosylation inhibitor tunicamycin

(Tm) than wildtype plants and gpa1-4 plants with a null mutation in the gene encoding

the α subunit of the heterotrimeric G protein. Leaves of agb1-2 mutant plants exhibit

markedly less cell death after Tm treatment than those of wildtype plants. The

transcriptional response of agb1-2 mutant plants to Tm is less pronounced than that of

wildtype plants. On the other hand, AtrbohD (AtrbohD null mutation) and AtrbohD/F

(AtrbohD and AtrbohF null mutation) mutant plants show more cell death in response to

treatment with Tm than wildtype plants and AtrbohF (AtrbohF null mutation) mutant

plants. The protective role of the endogenous ROS elicited by Tm treatment against

Tm-induced cell death is also seen using ROS scavengers. Moreover, the transcriptional

responses are delayed by the pretreatment with ROS scavengers.

A majority of the Arabidopsis Gβ protein is associated with the endoplasmic

reticulum (ER) and is degraded after Tm treatment, while Gα protein is not. Collectively,

these observations indicate that the Gβγ complex, not Gα, plays an important role in cell

death associated with the unfolded protein response in Arabidopsis. Moreover these

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observations imply a direct role for both ROS and G protein signaling in the UPR.

To elucidate the mechanism of G protein signaling transduction, we characterized

the Arabidopsis heterotrimeric G protein complex. Using fluorescence resonance energy

transfer (FRET) and co-immunoprecipitation, we show interaction between the β1 and γ

subunits and between the α1 and β1γ subunits of the Arabidopsis heterotrimeric G

proteins in plant protoplasts. We report that both the Gα and the Gβ subunits are

associated with macromolecular complexes in plasma membrane fractions. We estimate

the size of the G-protein-containing plasma membrane complex to be approximately 669

kD based on blue native polyacrylamide gel electrophoresis, suggesting that the

heterotrimeric G protein is present in a complex with other proteins. We find that Gα1 is

present in both the large ca. 669 kD complex and in smaller complexes in plants

homozygous for the agb1-2 Gβ1 null allele. Deletion of the Gβ1 interacting domain in

Gα1, ahGα1, abolishes normal function of Gα1 in the oxidative stress response to ozone,

suggesting the interaction between Gα1 and Gβ1γ complex may be required for

transmitting the O3 signal in plant cells. Treatment of the plasma membrane fraction with

hydrogen peroxide (H2O2), an important signaling molecule, results in the partial

dissociation of the Gα1 complex. This observation suggests direct or indirect activation of

G protein signaling by ROS in the oxidative stress response.

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Table of Contents

LIST OF FIGURES...............................................................................viii

ACKNOWLEDGEMENTS………………………………………………x

Chapter 1 INTRODUCTION

Overview of the Arabidopsis Heterotrimeric G Protein Genes and Mutants....1

Redox Signaling in the Arabidopsis Oxidative Stress Responses to ozone……10

Unfolded Protein Responses in Arabidopsis.........................................................17

References…………………………………………………………………….…..25

Chapter 2 DIFFERENT SIGNALING AND CELL DEATH ROLES

OF HETEROTRIMERIC G PROTEIN OF Α AND Β SUBUNITS

IN THE ARABIDOPSIS OXIDATIVE STRESS RESPONSE TO

OZONE

Introduction……………………………………………………………………..38

Material sand Methods………………………………………………………….40

Results…………………………………………………………………………....44

Discussion………………………………………………………………………...51

References………………………………………………………………………...54

Chapter 3 BOTH HETEROTRIMERIC G PROTEIN SIGNALING

AND ROS SIGNALING ARE IMPLICATED IN THE

ARABIDOPSIS UNFOLDED PROTEIN RESPONSE

vi

Introduction………………………………………………………………………58

Materialsand Methods…………………………………………………………...61

Results…………………………………………………………………………….66

Discussion………………………………………………………………………...78

References………………………………………………………………………...84

Chapter 4 CHARACTERIZATION OF THE ARABIDOPSIS

HETEROTRIMERIC G PROTEIN COMPLEX

Introduction………………………………………………………………………90

Materials and Methods…………………………………………………………..93

Results…………………………………………………………………………...100

Discussion……………………………………………………………………….112

References……………………………………………………………………….118

Chapter 4 CONCLUSION

Function of Heterotrimeric G Protein in Arabidopsis…………………………123

Heterotrimeric G Protein Complex in Arabidopsis……………………………125

Significance of the Present Findings ……………………………………….......126

Future Direction…………………………………………………………………129

References………………………………………………………………………..131

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LIST OF FIGURES

Figure 1. Altered ozone sensitivity in G protein mutant plants…………………………45

Figure 2. Different expression profiles of GPA1 and AGB1 genes

after ozone treatment………………………………………………………….47

Figure 3. Perturbed O3–induced oxidative burst in G protein mutants………………….48

Figure 4. Enhance resistance to O3–damage by lower does O3 pretreatment…………...50

Figure 5. Plants homozygous for null mutations in genes coding for the α and β subunits

of the heterotrimeric G protein exhibit different sensitivities to Tm induced

growth inhibition………………………………………………………………67

Figure 6. Leaf senescence and cell death in wildtype and agb1-2 mutant plants………..68

Figure 7. The Tm-induced UPR is attenuated in agb1-2 mutnat plant…………………..71

Figure 8. Tm induceds an oxidative burst and DPI delays UPR marker

gene expression………………………………………………………………..72

Figure 9. Suppressing ROS production increases Tm-induced cell damage and death…73

Figure 10. Tm-induced cell death is accelerated in atrbohD, atrbohF atrbohD/F………75

Figure 11. Gβ protein is localized in the ER and co-fractons with Bip………………….77

Figure 12. Gβ protein is degraded during the UPR……………………………………...78

Figure 13. Spectral characterics and cellular localization of CFP and YFP of Arabidopsis

G protein subunits…………………………………………………………...100

Figure 14. FRET images of CFP and YFP of Arabidopsis G protein subunits………... 104

viii

Figure 15. Detection of heterotrimeric G protein complexes in Arabidopsis………….107

Figure 16. Phenotypes of 4 weeks-old light-grown plants of the indicated

genetic contribution…………………………………………………………110

Figure17. Redox regulation of heterotrimeric G protein complex in Arabidopsis…….111

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ACKNOWLEDGEMENTS

This work has been conducted under the supervision of Dr. Nina Fedoroff. I have

been confronted with many chanllenges during my project. Eventually I was successful

with my defense and thesis. This would not be possible without the invaluable advice and

much-needed encouragement as well as continuing support from Dr. Feodoroff. As my

mentor, Dr. Fedoroff has greatly improved my critical thinking skills. As a real scientist,

Dr. Fedoroff has motivated me with strong scientific enthusiasim. Undoubtedly, the

training under her guidence has set a new stage for me and the research experiences in

her lab will continuously befenit my career as a scientist. I am deeply indebted to her for

giving me the study and work in her laboratory.

I also would like to express my gratitude to Dr. Sarah Assmann, Dr. David S.

Gilmour, Dr. Teh-hui Kao, Dr. Ming Tien for their service on my committee and

invaluable suggestions. I am particular thankful to my special committee number Dr.

Alan M. Jones from University of North Carolina at Chapel Hill for his willingness to

serve on my committee.

My special thanks go to my husband, Shijie Cai, for his understanding and

unconditional support of my study. He gave me lovely company during the last six years.

He’d rather stay with than going out for a funcy job. He has surely sacrificed a lot for me

and certainly deserves to share all the credits I have earnd so far.

x

Chapter 1

INTRODUCTION

Heterotrimeric G proteins are recognized as important signal transduction molecules

in all eukaryotic organisms from yeasts to humans (Neer, 1995). Compared to mammals

and invertebrates, simplicity of G protein gene family in Arabidopsis provides a unique

advantage for investigating its functions in various aspects of different physiological

processes (Jones and Assmann, 2004; Assmann, 2005a). Extensive studies in the last 10

years have shown that the Arabidopsis heterotrimeric G protein is involved in transducing

a variety of signals, including light, hormones, biotic and abiotic stressors

(Perfus-Barbeoch et al., 2004). However, the mechanism of G protein signaling

transduction in Arabidopsis is still under investigation (Assmann, 2005b).

Overview of the Arabidopsis Heterotrimeric G Protein Genes and Mutants

Heterotrimeric G Proteins in Mammals

G proteins are guanine-nucleotide binding proteins found in all eukaryotes

(Pennington, 1994). G proteins are classically divided into heterotrimeric G proteins and

small G proteins. Heterotrimeric G proteins consist of α, β and γ subunits, whereas small

G proteins are monomeric and similar in function to the α subunit of heterotrimeric G

1

proteins (Neer, 1995). In mammals, all three heterotrimeric G protein subunits belong to

multigene families (Wilkie and Yokoyama, 1994). At least 20 different Gα (Strathmann

and Simon, 1990), five or six Gβ (Seack et al., 1998) and 12 Gγ subunits (Cook et al.,

2001) have been identified in humans. All Gαs contain several important conserved

structural domains: a GTPase domain, a GTP binding domain and two switch domains,

which change conformation with binding of GTP or GDP (Bourne et al., 1991). The

important characteristic of Gβ subunits is a seven-bladed β-propeller structure (Lambright

et al., 1996; Sondek et al., 1996), which comprises seven or eight tandem repeats with a

conserved Trp-Asp (WD) domain. Gβ and Gγ subunits can interact strongly through the

coiled-coil structure formed between them (Sondek et al., 1996). All Gγs (Song and

Dohlman, 1996), as well as some species of Gα (Casey, 1994), can be modified by lipids

that attach proteins to membranes.

The classic heterotrimeric G protein functions as follows (Bourne et al., 1991). In the

inactive state, the GDP-bound α subunit associates with the Gβγ dimer to form a

heterotrimeric G protein complex. The heterotrimer interacts with a family of plasma

membrane receptors containing seven transmembrane domains (7-TM) known as G

protein coupled receptors (GPCRs). In metazoans, signal perception by the GPCR

catalyzes the exchange of GDP for GTP on an α subunit, causing Gα to dissociate from

the GPCR and Gβγ dimer. In the GTP-bound form, Gα regulates downstream target

proteins, as does the free Gβγ dimer. Gα has intrinsic GTPase activity and ultimately

causes Gα to return to the inactive state, where it can re-associate with Gβγ subunits. In a

2

few cases, the dissociation of Gα from Gβγ dimer is not necessary for the activation of G

protein signaling (Klein et al., 2000; Bunemann et al., 2003).

G protein activity can be manipulated using nonhydrolyzable GTP analogs and

ADP-ribosylating bacterial toxins. GTPγS is a nonhydrolyzable GTP analog that locks

mammalian Gα subunits in their active state. Conversely, GDPβS is a nonhydrolyzable

GDP analog that locks mammalian Gα subunits in their GDP-bound, inactive form. ADP

ribosylation by cholera toxin (CTX) modifies a conserved Arg residue found in the

mammalian Gα subunit, locking the protein into a conformation that inhibits its GTPase

activity, resulting in its prolonged activity (Gillison and Sharp, 1994). Pertussis toxin

(PTX) ribosylates a Cys with ADP near the carboxyl terminus of Gα and inactivates

signaling from GPCR to Gα (Carty, 1994). Mastoparan, a short peptide from wasp venom,

is able to activate G proteins by interfering with Gα-GPCR interactions, mimicking

GPCR activation (Klinker et al., 1994).

Heterotrimeric G Protein in Arabidopsis

In contrast to the large number of different G protein subunits in animals, Arabidopsis

has only one canonical Gα protein (AtGPA1) (Ma et al., 1990), one canonical Gβ protein

(AtAGB1) (Weiss et al., 1994), and two Gγ proteins (AtAGG1 and AtAGG2) (Mason and

Botella, 2000, 2001). All of these subunits have limited homology with their animal

counterparts. The AtGPA1 protein shows roughly 30% identity with the mammalian Gα

subfamily protein (Ma et al., 1990). It possesses the sites for CTX, N-myristoylation and

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N-palmitoylation but lacks the site for PTX. Although the biochemical properties of the

Arabidopsis AtGPA1 protein have not been characterized as extensively as those of the

rice GPA1 (RGA1) (Seo et al., 1997), recombinant AtGPA1 has been shown to bind

GTPγS and have GTPase activity, which can be enhanced by the regulator of G protein

signaling (AtRGS1) (Chen et al., 2003). AtAGB1 has about 42% similarity to mammalian

Gβ subunits (Weiss et al., 1994), and AtAGGs display 24%-31% identity with certain

mammalian Gγ subunits. Both AGG1 and AGG2 proteins contain C-terminal CaaX

motifs for farnesylation and geranylgeranylation (Mason and Botella, 2000, 2001). At the

cellular level, Gα1 has been immunolocalized in the plasma membrane and endoplasmic

reticulum (Weiss et al., 1997), while Gβ1 has been detected in the plasma membrane,

endoplasmic reticulum and Golgi structure (Obrdlik et al., 2000; Wang et al., 2007). Gγ1

and Gγ2 are both observed in the plasma membrane using fluorescent fusion proteins in

cowpea protoplasts (Adjobo-Hermans et al., 2006).

Structural modeling of Gα1, Gβ1, and Gγ1 shows that the functionally important

regions of the G protein heterotrimer structure are highly conserved in the Arabidopsis

subunits (Ullah et al., 2003), suggesting that they can form a heterotrimer similar to that

formed by the mammalian G protein subunits. Interactions between Gβ1, Gγ1 and Gγ2

have been detected by yeast two-hybrid and in vitro binding assays (Mason and Botella,

2000, 2001), and there is similar evidence from yeast two-hybrid and

co-immunoprecipitation assays for interactions between the rice Gα subunit and Gβ

subunits (Kato et al., 2004). The heterotrimerization of Arabidopsis G protein subunits in

4

cowpea protoplasts was recently reported.

Whole genome sequences of Arabidopsis are available. Mutant alleles of AtGPA1

(Ullah et al., 2001; Wang et al., 2001; Ullah et al., 2003) and AtAGB1 (Lease et al., 2001;

Ullah et al., 2003) by screening a T-DNA insertion library or a genetic screen of ethyl

methanesulfonic acid (EMS)-mutagenized plants were identified, which all facilitates the

functional study of the heterotrimeric G protein in Arabidopsis.

Important Roles of Heterotrimeric G Protein in Arabidopsis

Although pharmacological experiments suggested that plant heterotrimeric G proteins

mediate diverse signals in plants such as blue light (Warpeha et al., 1991), red light

(Romero et al., 1991), auxin, and abscisic acid (ABA) (Ritchie and Gilroy, 2000), recent

genetic studies provide direct evidence that Arabidopsis heterotrimeric G protein is

involved in the transmission of different kinds of signals, including hormones, biotic and

abiotic stress.

AtGPA1 and AtAGB1 mutant plants show both similar and opposite phenotypes at

various developmental stages (Ullah et al., 2003). Both mutants have rounded lamina in

their leaves and reduced cell division in their hypocotyls. Compared with those of

wild-type plants, fruit and seed weights are greater for both mutants. However, gpa1-1

sepals are longer than wild-type sepals, whereas agb1-1 sepals are shorter than wild-type.

gpa1-1 seedlings are smaller than wild-type, whereas agb1-1 seedlings are larger than

wild-type. agb1-1 and agb1-2 have more roots mass than wild-type, whereas gpa1-1 and

5

gpa1-2 have less root mass than wild-type. Moreover, agb1-1 mutant plants have

increased apical dominance and gpa1-1 mutant plants have decreased apical dominance.

There are also some phenotypes unique to each mutant. Pedicels in gpa1-1 mutant are

uniquely long and agb1-1 mutant leaves are uniquely curly. The divergent alterations in

developmental phenotypes of G protein mutants suggest that functions of Arabidopsis Gα

and Gβγ can differ in specific cell types and different developmental processes (Ullah et

al., 2003).

Likewise, phenotypes are altered differentially when G protein mutants are exposed to

varieties of hormones, biotic and abiotic stresses. gpa1-1 and gpa1-2 mutant seeds are not

only less sensitive to GA, but completely insensitive to brassinosteroids (BR) (Ullah et al.,

2002). However, the gpa1-4 mutant shows moderately enhanced sensitivity to ABA

inhibition in seed germination (Ullah et al., 2002; Pandey and Assmann, 2004), while the

agb1-2 mutant exhibits hypersensitivity to ABA inhibition in seed germination. Moreover,

only the agb1-2 mutant exhibits decreased sensitivity in a number of jasmonic acid

(JA)-induced responses such as inhibition of root elongation and seed germination

(Trusov et al., 2006). In biotic stress responses, agb1-1 shows the enhanced susceptibility

to the fungal pathogen Plectosphaerella cucumerina, and necrotrophic pathogens

Alternaria brassicicola and Fusarium oxysporum, whereas the gpa1-4 mutant shows

enhanced resistance to these pathogens compared to wild-type (Llorente et al., 2005). In

abiotic stress responses, the agb1-2 and agb1-1 mutants are more sensitive to O3 damage

than wild-type plants, whereas the gpa1-4 mutant is more resistant to O3 damage than

6

wild-type (Joo et al., 2005). Moreover, only the agb1-2 mutant shows a more resistant

phenotype to tunicamycin (Tm, protein glycosylation inhibitor)-induced leaf cell death

(Wang et al., 2007). This summary of G protein mutant phenotypes in response to

hormones and stressors pinpoints the important roles of the Arabidopsis heterotrimeric G

protein in a variety of signal transduction events.

The functions of Arabidopsis Gα and Gβγ are well understood in some biological

processes. Ozone induces a bimodal oxidative burst (reactive oxygen species, ROS) in

wild-type plants (Joo et al., 2005). The first peak is almost entirely missing in both

gpa1-4 and agb1-2 mutant plants. The late peak is normal in agb1-2 but missing in

gpa1-4 mutant plants. This observation indicates that only Gα is required for the

production of the late peak, but both Gα and Gβγ, or the entire heterotrimeric G protein,

are required for the first peak. Further characterization of ROS production induced by

ozone shows that the production of ROS in the late peak is mainly attributable to its

ability to activate the NADPH oxidase signaling pathway, which is intact in agb1-2, but

not in gpa1-4 mutant plants. This result indicates that Gα subunit can mediate activation

of membrane-bound NADPH oxidases in Gβ protein independent of the formation of the

heterotrimer.

The Arabidopsis Gα and Gβγ proteins have also been reported to play different

roles in modulating cell division in roots (Chen et al., 2006). By comparing root growth

rate and lateral root formation in gpa1-4 and agb1-2 single and double mutants as well as

in transgenic lines overexpressing AtGPA1 in agb1-2 and overexpressing AtAGB1 in

7

gpa1-4 mutant backgrounds, the functions of Gα, Gβγ and heterotrimeric G protein in

cell proliferation in roots were determined. Results show that the heterotrimeric complex

is a negative regulator of cell proliferation in root growth, whereas the GTP-bound Gα

subunit is a positive regulator in this process. On the other hand, Gβγ dimer has a

function independent of Gα in attenuating cell division during the formation of lateral

roots. These comparisons also clearly suggest that Arabidopsis Gα and Gβγ have both

common and independent roles in the modulation of cell division in roots.

Proteins that Interact with the Arabidopsis Heterotrimeric G Protein

The different functions of Arabidopsis Gα or Gβγ may result from their interaction

with different proteins, which perform functions in a variety of development-related

events and stress-associated processes in response to G protein signals. A number of

Arabidopsis Gα-interacting proteins have been identified, although no downstream

targets of Gβγ signaling have yet been identified in plants. By yeast two-hybrid analysis

and in vitro pulldown assays, AtPirin1, a presumed transcription factor, and Gα1 have

been shown to physically interact. An atpirin1 null mutant is hypersensitive to ABA in

inhibition of seed germination and early seedling development (Lapik and Kaufman,

2003). Given the enhanced sensitivity of gpa1-1 and gpa1-2 mutants to ABA in inhibition

of seed germination (Pandey et al., 2006) and the interaction between AtPirin1 and Gα1, it

was inferred that the enhanced sensitivity of gpa1-4 mutant to ABA could be due to loss

of an activating signal from Gα1 to AtPirin1, thought to be a negative regulator of ABA

8

action (Assmann, 2005b).

Another well-known Gα1 interacting protein is Arabidopsis phospholipase Dα1

(AtPLDα1). Gα1 was shown to interact with AtPLDα1 using pulldown assays in bacteria

and plant extracts (Zhao and Wang, 2004). The DRY motif in AtPLDα1, which is

conserved in G-protein coupled receptors, was identified as the interaction site with Gα1

using site-directed mutagenesis (Zhao and Wang, 2004). The biological significance of

their interactions was determined by introducing a mutant PLDα1 (AtPLDα1k564A), which

decreases the binding of Gα1 to PLα1 by 90%, into the PLDα1 mutant plants. The result

shows that AtPLDα1k564A can’t replace the function of PLDα1 in the ABA response to

inhibit stomatal opening, suggesting that Gα1 acts downstream of PLDα1 and

phosphatidic acid (PA) to regulate the ABA inhibitory effect on stomatal opening (Mishra

et al., 2006). Additional components of G protein signaling have also been identified as

Gα1 interacting proteins such as AtRGS1 (regulator of G protein signaling) (Chen et al.,

2003), AtGCR1 (G protein coupled receptor 1) (Chen et al., 2004; Pandey and Assmann,

2004), AtGCR2 (G protein coupled receptor 2) (Liu et al., 2007). AtRGS1 accelerates the

GTPase activity of Gα1 and acts as a negative regulator of Gα1 in modulating cell

proliferation in roots and hypocotyls (Chen et al., 2003). AtGCR1 and AtGCR2 both have

a predicted 7 transmembrane (TM) structure, which is conserved in mammalian GPCRs

(Pandey and Assmann, 2004; Liu et al., 2007). Although they share similar structural

properties, they play different regulatory roles in Gα1 functions. AtGCR1 acts in concert

with Gα1 and Gβ1 in ABA signaling during germination and early seedling development

9

(Pandey et al., 2006), whereas AtGCR2 has an opposite effect on this process. AtGCR2

and Gα1 function together to mediate ABA signal in guard cells, whereas AtGCR1 may

act as a negative regulator in this response. Since a large pool of predicted 7TM

containing proteins is present in Arabidopsis, it is likely that more AtGCRs could be

identified (Pandey and Assmann, 2004; Liu et al., 2007). The more components in the G

protein signaling pathway that are identified, the more complicated are the emerging

picture of G protein regulatory mechanisms in Arabidopsis. Nevertheless, these efforts

will eventually contribute to dissecting the mechanism of G protein signaling

transduction.

Redox Signaling in the Arabidopsis Oxidative Stress Response to Ozone

Ozone Effects on Plant Defense

Ozone (O3) has been recognized as a major component of photochemical air pollution

responsible for causing significant damage in both nature and cultivated plants since 1958

(Darley et al., 1959). O3 generally inhibits plant photosynthesis and growth to influence

the likelihood of biotic plant disease (Ordin, 1965; Coulson and Heath, 1974). O3 is taken

up from leaf stomata and is apparently destroyed rapidly in the apoplast compartment.

Ozone dissociates in aqueous solutions and produces reactive oxygen species (ROS) such

as superoxide (O2-) and hydrogen peroxide (H2O2), which can further react with transition

metals generating hydroxyl radicals (OH.) and singlet oxygen (Kanofsky and Sima, 1995).

10

Superoxide can also react with amine, phenolic compounds and extracellular ascorbate to

exacerbate the production of hydroxyl radicals (Kanofsky and Sima, 1995). These ROS,

especially singlet oxygen and hydroxyl radicals, are harmful to all living organisms due

to their strong oxidizing effects on biologically important macromolecules. Thus, O3 is

widely used to study the plant’s defense response to the oxidative stress.

O3 triggers plant defense responses including the production of an oxidative burst,

the biosynthesis of antimicrobial compounds, cell wall proteins, antioxidants, and

signaling molecules, as well as the apoptotic hypersensitive response (HR) and the

systemic acquired resistance (SAR) (Mahalingam et al., 2003). The plant’s response to

this oxidative stress shares some commonalties with the pathogen defense response,

including the production of ROS and induction of the HR and SAR (Lamb and Dixon,

1994; Lamb and Dixon, 1997; Baker and Orlandi, 1999; Inze and Van Montagu, 2002;

Scheel, 2002; Apel and Hirt, 2004).

Production of the Oxidative Burst

A variety of biotic and abiotic stressors trigger a transient increase in endogenous

reactive oxygen species, predominantly O2- and H2O2 in plant cells (Levine et al., 1994;

Schraudner et al., 1998; Joo et al., 2005). This phenomenon is termed “oxidative burst”.

The oxidative burst was first reported in plants challenged with an incompatible pathogen

Phytophthora infestans. Subsequently, the oxidative burst has also been observed in

plants challenged with fungal, bacterial and viral pathogens, as well as with many other

11

abiotic stressors including high temperature, intense light, drought, cold and air pollutants

such as O3. In most cases, the plant oxidative burst is a biphasic response, comprising a

primary peak 1-2h, followed by a secondary peak with greater magnitude 3-6 h after

infection (Lamb and Dixon, 1994). However, the occurrence, intensity and duration of

the oxidative burst in plants vary depending on the plant system studied and the elicitor

used (Allan and Fluhr, 1997; Lamb and Dixon, 1997; Tenhaken and Rubel, 1998). The

oxidative burst triggered by ozone is also dependent on treated plants, intensity and

duration of ozone (Schraudner et al., 1998; Joo et al., 2005).

Detailed studies on ROS production in response to different stressors have shown that

the ROS can be distinguished enzymatically and spatially (Suharsono et al., 2002; Apel

and Hirt, 2004; Joo et al., 2005; Fedoroff, 2006). There are many enzymatic sources of

ROS in plants, both extra- and intracellular, including varieties of oxidases and

peroxidases, such as cell-wall peroxidases and amine oxidases, plasma membrane-bound

NADPH oxidases, and intracellular oxidases and peroxidases in mitochondria,

chloroplasts, peroxisomes and nuclei (Foyer and Noctor, 2005). There are also various

metabolic pathways to continuously produce ROS as byproducts in different cellular

compartments. While different mechanisms have been proposed for the ROS increase

triggered by either biotic or abiotic stressors, how various cellular ROS sources are

activated and propagated to produce the transient burst is not well understood (Apel and

Hirt, 2004; Fedoroff, 2006).

Recently we reported that Arabidopsis heterotrimeric G protein signaling is required

12

to activate the intracellular sources of ROS that contribute to the first component of the

biphasic, O3-elicited oxidative burst (Joo et al., 2005). ROS are produced rapidly in guard

cell chloroplasts and peripheral membranes after O3 treatment and these two sources of

ROS act as signals to elicit production of more ROS in the adjacent cells, resulting in the

first oxidative burst. However, this oxidative burst is absent in both gpa1-4 and agb1-2

mutant plants. This result further suggests that ROS production in response to O3 may

require not only ROS-generating enzymes but other signaling molecules to finely control

the initiation of ROS production. The small GTPase, one of monomeric G proeins, has

been implicated in the activation of membrane bound NADPH oxidase in the pathogen

response in rice (Suharsono et al., 2002).

Signaling Roles of the Oxidative Burst

The hazards of reactive oxygen species have long been recognized, although the

essential roles for them in signaling are still under investigation (Sauer et al., 2001;

Cormack et al., 2002; Droge, 2002). Nevertheless, increasing evidence shows that ROS

are produced locally or systemically and act specifically for signaling in both plant stress

responses and developmental processes (Joo et al., 2001; Schopfer et al., 2002; Overmyer

et al., 2003). The oxidative burst can induce programmed cell death (PCD) in various

systems. Genetic studies support this concept by showing that Arabidopsis atrbohD or

atrbohF mutant plants lacking functional rbohD or rbohF genes (i.e., respiratory burst

oxidase homolog genes) exhibit reduced ROS generation and PCD following bacterial

13

challenge (Torres et al., 2002). ABA stimulates ROS production, which induces stomatal

closure via activation of plasma membrane calcium channels (Kwak et al., 2003).

Moreover, stomatal closure and plasma membrane calcium channel activation are reduced

in atrbohD atrbohF double mutants but can be restored with H2O2, suggesting that ROS

serve as second messengers in the ABA response in Arabidopsis guard cells (Kwak et al.,

2003). H2O2 is also involved in auxin signaling and gravitropism in maize roots (Joo et al.,

2001). Arabidopsis atrbohC mutant plants lacking a functional rbohC gene show a root

development defect phenotype, suggesting that ROS function as local signaling

molecules in Arabidopsis root development (Carol et al., 2005; Jones et al., 2007).

ROS react with proteins directly in many different ways, such as oxidizing thiol

residues like Cys, Met, attacking Lys, Pro, Arg and Thr to result in the formation of

protein carbonyl derivatives (Johansson et al., 2004). Thus, ROS act on some ROS sensor

protein, transcription factors and components of signaling pathways to change their

conformation and regulate their functions, suggesting that ROS serve as signaling

molecules (Fedoroff, 2006). ABI1 is a member of protein phosphatase 2C family (PP2C)

in Arabidopsis and plays a negative role in ABA signaling (Leung et al., 1997). An

enzymological study shows that H2O2 has a strong inactivating effect on ABI1 activity,

which may be caused by oxidation of cysteine residues in the active site (Meinhard and

Grill, 2001; Meinhard et al., 2002). Thus H2O2, as a second messenger, could mediate

ABA signaling by inactivating the negative regulator ABI1. Similarly, Arabidopsis

protein tyrosine phosphatase (AtPTP), which de-phosphorylates AtMAPK6, can also be

14

inactivated by H2O2 (Gupta and Luan, 2003). Thus H2O2 activates AtMAPK6 and

concomitantly inhibits the activity of AtPTP (Moon et al., 2003).

Unlike OxyR protein in bacteria and Gpx3 protein in yeast, ROS sensor proteins have

not yet been identified in Arabidopsis (Zheng et al., 1998; Lee et al., 2004). However, an

Arabidopsis protein, AtOXS2, has been reported to have properties of an ROS sensor

(Branvillain et al., 2006). Yeast cells expressing AtOXS2 cDNA show an enhanced ability

to tolerate diamide-induced oxidative stress. AtOXS2 protein belongs to a zinc-finger

transcription factor family but with C-terminal nuclear export sequence (NES). Under

non-stress condition, AtOXS2 protein stays in the cytoplasm, whereas under stress

conditions, the protein moves into the nucleus and activates flowering gene expression to

promote early reproduction. Although how this shuttling mechanism is triggered by stress

is not clear yet, it is likely that its nuclear export sequence is shielded or modified by

ROS produced in response to stress (Branvillain et al., 2006).

The transcriptional regulatory factor NPR1 (Nonexpressor of Pathogenesis-Related

genes) is activated in a redox regulated manner (Pieterse and Van Loon, 2004). Under

unstressed conditions, NPR1 is locked in the cytoplasm in disulfide-bonded

intermolecular oligomeric form. With the application of salicylic acid (SA), the

intermolecular disulfide bonds are reduced, resulting in the release of monomeric NPR1,

which can move into the nucleus and activate expression of defense genes such as PR

(Rairdan and Delaney, 2002). NPR1 becomes constitutively monomeric and active for

promoting defense gene expression under unstressed condition when it is mutated at the

15

Cys82 or Cys216 residues (Despres et al., 2003; Moon et al., 2003). SA, as a mandatory

hormone of the SAR, can induce ROS production, which in turn can enhance Cu-Zu

superoxide dismutase (SOD), peroxidase, glutathione reductase and ascorbate peroxidase

activities or their transcript level in plants to counteract the toxic effects of ROS

(Mahalingam et al., 2003). As a result of the accumulation of antioxidants, oligomeric

NPR1 could be reduced and activated to be monomeric NPR1 (Fobert and Despres,

2005).

In mammals, some signaling pathways activated by H2O2 show similarities with those

activated by Gi protein coupled receptors. For example, two mammalian Gα proteins, Gαi

and Gαo, are ROS targets and directly regulated by H2O2 (Nishida et al., 2000). H2O2

treatment promotes increased affinity of GTP for the Gα subunit in dose-dependent

manner and activates Gβγ signaling as well as its downstream effectors (Nishida et al.,

2000). Moreover, Cys287 in Gαi and Gαo required for their ROS activation (Nishida et al.,

2002) is conserved in AtGα1 (Ma et al., 1990). This raises the intriguing possibility that

Arabidopsis G protein activity is redox regulated.

ROS can also play its signaling role in an indirect way (Orozco-Cardenas and Ryan,

1999). ROS induces production of the intercellular messengers such as salicilic acid (SA),

jasmonic acid (JA) and ethylene, which act in concert to regulate O3-induced protective

and adaptive responses such as defense gene expression and hypersensitive cell death

(Sharma et al., 1996; Klessig et al., 2000; Orozco-Cardenas et al., 2001; Tamaoki et al.,

2003). Since these messengers have a longer life than ROS, some function of them may

16

be beyond the direct control of ROS in O3 response (Apel and Hirt, 2004). Genetic

studies about mutant plants lacking SA, JA or ethylene signaling have established their

essential roles in O3 response (Nawrath and Metraux, 1999; Rao and Davis, 1999; Rate et

al., 1999; Morris et al., 2000; Overmyer et al., 2000; Shah et al., 2001). However, their

interconnected relationship with ROS is still not well understood (Torres et al., 2005).

The ROS production can alter the redox environment in cells, which itself can cause

numbers of biological responses like modulating enzyme activity and altering gene

expression level, especially in chloroplasts (Creissen et al., 1999; Morris et al., 2000).

Although redox regulatory processes are emerging to be central for activating the stress

responses, given the multiple sources of ROS and the complexity of cell redox

environments, identification of ROS target and ROS action is still challenging for the

future research (Fedoroff, 2006).

Unfolded Protein Responses in Arabidopsis

ER Stress and Unfolded Protein Responses in Mammals

The endoplasmic reticulum (ER) is a primary organelle in which secretory proteins

are synthesized, modified, and delivered to their target sites (Schroder and Kaufman,

2005). ER stress occurs when protein folding, modification and transportation are

perturbed (Schroder and Kaufman, 2005). To protect cells from this stress, a series of

signaling cascade, termed the unfolded protein response (UPR), is activated to re-balance

17

the folding demand and the folding capacity in the ER (Wu and Kaufman, 2006).

UPR can be induced experimentally and physiologically (Rutkowski and Kaufman,

2004). Chemicals which prevent proteins from folding properly can activate UPR.

Tunicamycin, which inhibits N-linked glycosylation in the ER (Duksin and Mahoney,

1982), thapsigargin (Tg), which depletes the cell’s energy stores for protein folding

(Thastrup et al., 1990) and dithiothreitol (DTT), which creates reductive stress in ER

(Jamsa et al., 1994), are all widely used in ER stress and UPR studies in mammalian cells.

Some physiological responses can also activate the UPR including certain pathogen

infections, such as hepatitis C, differentiation of secretory cells and nutrient deprivation

(Rutkowski and Kaufman, 2004).

To increase the capacity of protein folding and the disposal of misfolded proteins in

the ER, the array of biochemical and physiological processes believed to be activated in

the UPR includes 1) induction of ER resident chaperone synthesis, 2) attenuation of

translation of most proteins, 3) up-regulation of ER-associated degradation pathways

(ERAD), and 4) initiation of apoptotic cell death, which occurs when the three

prosurvival responses have failed (Wu and Kaufman, 2006). Perturbation of any of

these processes can cause cells or organisms to be susceptible to the ER stress.

Furthermore, the ER stress is thought to be involved in certain diseases such as diabetes,

Alzheimer’s, atherosclerosis, and cancer (Zhao and Ackerman, 2006).

In mammalian cells, the three distinct protective responses of the UPR are believed

to be mediated by ER-resident IRE1 (inositol-requiring transmembrane kinase and

18

endonuclease 1), ATF6 (activation of transcription factor 6) and PERK (protein

kinase–like ER kinase) respectively (Bertolotti et al., 2000; Liu et al., 2000). These

three proteins remain bound by ER-resident luminal binding protein (BiP) under resting

conditions. When the ER is stressed, BiP preferentially binds to the unfolded proteins,

effectively being titrated away from IRE1, ATF6 and PERK (Bertolotti et al., 2000).

The dissociation from BiP activates PERK, which phosphorylates the translation

elongation factor eIF2α and furthermore inhibits translation of new proteins (Harding et

al., 1999). After the dissociation from BiP, ATF6 is translocated to the Golgi and

cleaved by S1/S2 protease to form the active ATF6, which is a transcription factor

promoting chaperone gene expression (Li et al., 2000; Wang et al., 2000). Upon its

release from BiP, IRE is activated by dimerization and auto phosphorylation. The active

IRE cleaves the precursor of XBP1u mRNA to mature XBP1s mRNA encoding X-box

binding protein, by the effective translation which enters the nucleus and functions as a

transcription factor inducing the expression of genes involved in unfolded protein

degradation (Yoshida et al., 2001).

Compared with these adaptive pathways, apoptotic cell death induced by the ER

stress is less well understood (Schroder and Kaufman, 2005). Apoptosis is mainly

initiated by two pathways. One is an intrinsic pathway; the other is an extrinsic pathway

(Rutkowski and Kaufman, 2004). The intrinsic pathway is activated by intracellular

stresses like DNA damages. The extrinsic pathway is activated by extracellular stimuli

and mediated by cell surface receptors. The ER stress is more like an intrinsic than an

19

extrinsic apoptotic signal, although components of both pathways appear to be involved

in ER-induced apoptotic cell death.

The difference in the concentration of Ca2+ between the ER, cytosol, and

mitochondria is thought to control the initiation of apoptotic cell death (Boya et al.,

2002; Nguyen et al., 2002). Transient elevation of the cytosolic Ca2+ concentration

([Ca+2]cyt) released from the ER by the insertion of BH family proteins like Bak and

Bax into the ER membrane activates the Ca2+-dependent caspase calpain (Breckenridge

et al., 2003; Reimertz et al., 2003). Activated calpain cleaves and activates procaspase-9,

which eventually activates the executioner caspase, caspase-3. Mitochondria can also

take up Ca2+ released from the ER, triggering cytochrome c release into the cytoplasm

and formation of the apoptosome complex. The apoptosome can activate caspase-9,

which activates procaspase-3 to initiate cell death (Crompton, 1999).

The ER stress can enhance the interaction between c-Jun N-terminal inhibitory

kinase (JIK) and tumor necrosis factor receptor-associated factor 2 (TRAF2), which

also interacts with IRE1α. Formation of this trimeric complex mediates the signal to

apoptosis signal-regulating kinase 1 (ASK1), which leads to cell death (Yoneda et al.,

2001; Nishitoh et al., 2002). This signaling cascade represents an extrinsic pathway of

apoptosis induced by the ER stress. Although some essential molecules in UPR induced

apoptotic pathways have been identified, the detailed molecular mechanism of the

cellular decision to commit cells to apoptosis in the UPR remains poorly understood

(Rutkowski and Kaufman, 2004; Schroder and Kaufman, 2005; Wu and Kaufman,

20

2006).

ER Stress and Unfolded Protein Responses in Arabidopsis

Although the study of the UPR and ER stress in plants is not extensive, it has been

shown that plants have a protective transcriptional response when exposed to ER stress,

including induction of genes encoding protein-folding enzyme and chaperones (Martinez

and Chrispeels, 2003; Kamauchi et al., 2005). Both tunicamycin (Tm), an inhibitor of

N-linked protein glycosylation, and CPA cyclopiazonic acid (CPA), an inhibitor of the

ER-type II Ca2+, can trigger the UPR and programmed cell death in cultured plant cells

(Zuppini et al., 2004). However, only a few orthologs of mammalian UPR proteins have

been identified so far in plant UPRs. These are Ire1 (Noh et al., 2002), eIF2α (Chang et

al., 1999; Chang et al., 2000) and p58IPK (Bilgin et al., 2003) . The Arabidopsis genome

has no orthologs of the ER stress-induced XBP1 or ATF6 genes, although other

transcription factors like bZIP60 (Iwata and Koizumi, 2005) and NTM1 (Kim et al., 2006)

contain a trans-membrane domain, which can be cleaved by a peptidase and hence may

function in similar manner as ATF6.

Recently we reported that ER stress induced by Tm can cause leaf senescence in

Arabidopsis (Wang et al., 2007). Senescence is the aging process in the last

developmental stage of plants (Gepstein, 2004). In mammalian cells, unfolded proteins

also accumulate with aging, thus it seems that the ER stress accelerates the aging of

plants. Senescence is defined as a specific type of programmed cell death (PCD) in plants.

21

As described above, the ER stress can activate PCD, which suggests that PCD induced by

the ER stress is conserved in plant and mammals. However, the mechanism of PCD in

plants is not as well understood as PCD in mammals.

It was reported that cyclopiazonic acid (CPA), an inhibitor of the ER-type II Ca2+

pump, induces cell death resembling PCD in cultured soybean cells (Zuppini et al., 2004).

Moreover, CPA also elicits the activity of a caspase 3-like protease. CPA-treated cultured

soybean cells exhibit a bi-phasic increase in [Ca+2]cyt, with an initial transient peak at

about 2 min after administration of CPA and a longer subsequent sustained rise in

[Ca+2]cyt, with a peak at about 10 min. Although the [Ca+2]cyt burst was demonstrated in

response to treatment by CPA, the causal connection between PCD and [Ca+2]cyt has not

been established yet. The intracellular Ca2+ chelator BAPTA-AM can not prevent cell

death caused by CPA.

Plants lack caspases, which play a central role in PCD in animal cells (Thornberry

and Lazebnik, 1998), but contain a family with 9 related proteins termed metacaspases,

which have a different cleavage specificity (Arg/Lys) than caspases (Vercammen et al.,

2004; Watanabe and Lam, 2005). Whether they contribute to ER stress-induced cell death

has not been determined, although it has been reported that the metacaspase mcII-Pa is

critical for PCD during Norway spruce embryogenesis (Bozhkov et al., 2005). Plants also

lack BH family protein like Bax and Bak, however, plants contain the Bax inhibitor-1

(BI-1), which can suppress the H2O2-induced apoptosis in Arabidopsis. Thus, it is likely

that BI-1 is involved in ER stress-induced cell death (Kawai et al., 1999; Sanchez et al.,

22

2000; Kawai-Yamada et al., 2001).

As important a signaling molecule as calcium (Sanders et al., 1999), ROS can play

critical roles in Tm mediated plant ER stress response. The ER strictly requires a

reducing environment for protein folding, disulfide bond formation and some

post-translational protein modifications (Fewell et al., 2001). Any disturbance of the

reducing environment will inhibit the activity of protein foldase and protein disulfide

isomerase (PDI), which will consequently result in the ER stress or aggravate the ER

stress (Fewell et al., 2001). On the other hand, the ER stress can disrupt the electron

transfer chain in protein disulfide bond formation, which results in the generation of ROS

(Noiva, 1999). This is how oxidative stress and ER stress are interconnected by redox

mechanisms.

ROS could act on chaperone proteins to regulate their biological activity. The heat

shock protein (hsp70), a eukaryotic chaperone, provides a remarkable example of a

delicate redox regulation mechanism for altering its chaperone activity upon oxidative

stress. Hsp70 can be oxidized by S-glutathionylation and the glutationylated form is more

effective in preventing protein aggregation than the reduced form (Hoppe et al., 2004;

Shelton et al., 2005). Heat shock factor (Hsf1) belongs to a highly conserved eukaryotic

transcription factor family, which induces the expression of a variety of heat shock

proteins in response to heat, oxidative stress and a variety of other stressors (Young et al.,

2004). The DNA binding activity of Hsf1 is directly promoted by oxidation with H2O2.

Arabidopsis contains both hsp70 and heat shock factors, some of which have been

23

identified as potentially redox-regulated (Ahn and Thiele, 2003).

ROS production is also involved in the UPR associated apoptotic pathway. CPA

elicits an oxidative burst at 25 minutes after treatment of soybean cells prior to the

occurrence of PCD (Zuppini et al., 2004). The relationship between the oxidative burst

and cell death in the UPR-associated apoptotic pathway remains unclear. Nonetheless,

Arabidopsis oxidase mutants like rbohD and rbohF together with the application of ROS

generation inhibitors and scavengers will help to provide insight into the role of ROS in

UPR-associated cell death.

24

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Chapter 2

DIFFERENT SIGNALING AND CELL DEATH ROLES OF HETEROTRIMERIC

G PROTEIN OF α AND β SUBUNITS IN THE ARABIDOPSIS OXIDATIVE

STRESS RESPONSE TO OZONE*

Abstract

Arabidopsis thaliana plants of homozygous gpa1-4 (Gα null mutation) are less

sensitive to O3 damage than wild-type Columbia-0 plants, whereas agb1-2 (Gβ null

mutation) are more sensitive to O3 damage than wild-type Columbia-0 plants. The genes

encoding the α and β subunits of the Arabidopsis heterotrimeric G protein are

differentially expressed in the course of the oxidative stress response to O3 and play

different roles in the plant’s response. The first peak of the biphasic oxidative burst

elicited by O3 in wild-type plants is absent in both mutant plants, suggesting that the first

peak of the oxidative burst requires both the α and β subunits or the intact heterotrimeric

G protein. The second peak is missing in gpa1-4 mutant plants, but is normal in agb1-2

mutant plants, suggesting that the second peak of the oxidative burst requires only the α

subunit but not the β subunit. Thus, the opposing O3 phenotypes and the differential ROS

production pattern between gpa1-4 and agb1-2 mutant plants indicate that the α and β

subunits play separable roles in cell death associated oxidative stress response to O3.

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* This part was contained in the Plant Cell Vol. 17, 957–970.

Introduction

Unlike yeast and mammals, plants have a small number of heterotrimeric G proteins

(Assmann, 2002). The Arabidopsis genome encodes a single canonical α (AtGPA1) (Ma

et al., 1990) and a single canonical β subunit (AtAGB1) (Weiss et al., 1994) and at most 2

γ subunits (AtAGG1 and AtAGG2) (Mason and Botella, 2000, 2001). Although there is

no experimental evidence to show the interaction among Arabidopsis G protein subunits,

structural predictions for Gα1, Gβ1, and Gγ1 suggest that they can form a heterotrimer

similar to that formed by the mammalian G protein subunits (Ullah et al., 2003). The

results of studies in Arabidopsis plants with null mutations in the genes encoding the α

and β subunits show that the plant heterotrimeric G protein is involved in the

transmission of different kinds of signals, including light, hormones and external stresses

(Perfus-Barbeoch et al., 2004). Some opposite phenotypes in phenotypic profiling of the

gpa1 and agb1 mutants suggest positive and negative regulatory roles for the α and

β subunits in different biological processes throughout development (Ullah et al., 2003).

Here we address the role of the heterotrimeric G protein in the oxidative stress response

to ozone (O3).

O3, a component of photochemical smog, represents an oxidative stress to living

organisms and is a major atmospheric pollutant, damaging crops and forests (Runeckles

et al., 1990). Like other stresses, O3 triggers a series of changes in plants to adapt to this

external stimulus, such as the production of reactive oxygen species (ROS), the

38

biosynthesis of antioxidants, cell wall proteins and signaling molecules, as well as the

hypersensitive response (HR) and systemic acquired resistance (SAR) (Lamb and Dixon,

1994; Levine et al., 1994; Lamb and Dixon, 1997; Baker and Orlandi, 1999; Droge, 2002;

Inze and Van Montagu, 2002). Plants prevent pathogens from invading and further

propagating from the infection site by activating a programmed cell death called HR

(Scheel, 2002). Signaling through the α subunit of the heterotrimeric G protein to activate

membrane-bound NADPH oxidase has been implicated in the development of disease

resistance and the apoptotic HR in rice (Suharsono et al., 2002).

A variety of biotic and abiotic stressors trigger a transient increase in endogenous

reactive oxygen species, predominantly superoxide and H2O2 in plant cells, termed

oxidative burst (Baker and Orlandi, 1999; Overmyer et al., 2003; Apel and Hirt, 2004). In

plants, ROS are mainly generated by enzymes and metabolic reactions in chloroplasts,

mitochondria and peroxisomes (Baker and Orlandi, 1999; Overmyer et al., 2003; Apel

and Hirt, 2004). In addition to that, oxidases and peroxidases in the apoplast and plasma

membrane have also been implicated in the ROS production during pathogen and ABA

responses (Torres et al., 2002; Kwak et al., 2003). The hazards of reactive oxygen species

have long been recognized, although the essential roles of them for signaling are still

under investigation. Evidence increasingly shows that ROS are essential for auxin and

ABA signaling, as well as in activating stress and defense responses (Lamb and Dixon,

1997; Joo et al., 2001; Sauer et al., 2001; Inze and Van Montagu, 2002). However, the

oxidative burst elicited by O3 has not been analyzed with respect to its cellular sources

39

and its role for O3 responses has not been determined yet (Schraudner et al., 1998; Rao

and Davis, 1999; Inze and Van Montagu, 2002; Mahalingam et al., 2003).

I show here that the α and β subunits of the Arabidopsis heterotrimeric G protein

play different roles in the plant’s response to O3. First, I show that plants lacking the α

subunit are more resistant to O3 damage than Col-0 plants, while plants lacking β subunit

are more sensitive to O3 damage than Col-0 plants. The genes encoding AtGPA1 and

AtAGB1 are differentially expressed during the time course of the O3 response. I show

that both Gα and β proteins are required for the first peak and only Gα is required for the

second peak of the biphasic oxidative burst elicited by O3.

Materials and Methods

Plant Materials, Growth Conditions, O3 Treatment

Gα null mutant plants (gpa1-4), Gβ null mutant (agb1-2) plants, and double mutant

plants (gpa1-4, agb1-2) in the Columbia background were kindly provided by Dr. Jones

(Ullah et al., 2003). Plants were grown in MetroMix 200 (Scotts-Sierra Horticultural

Products Company, Marysville, OH) in 5 cm pots (50 per flat) at 65% humidity under

fluorescent light at 30 W/m2/s with a 14 h light/10 h dark photoperiod for 4-4.5 weeks.

For O3 treatments, 4 week-old plants were transferred to the O3 fumigation chamber and

exposed to 350± 50 ppb O3 for 6 hrs or to 500±50 ppb or 700±50 ppb ozone for 3 hrs.

Ozone was generated using an Ozone Generator (Model No. 2000, Jelight Company,

40

Inc.), and monitored with an Ozone Monitor (Model 450, Advanced Pollution

Instrumentation, Inc., San Diego, CA). Control plants were transferred to an adjacent

chamber under identical growth conditions except for the O3 treatment. For pretreatment

experiments, 4 week-old plants were transferred to the O3 fumigation chamber and

exposed to 350± 50 ppb O3 for 2 hrs; then were taken out to an adjacent chamber without

O3 for 2 hrs and finally transferred to the O3 fumigation chamber and exposed to 700±50

ppb ozone for 3 hrs. Entire rosettes were harvested at different times after ozone treatment

by cutting them from the roots and freezing them at -80°C for RNA analysis and ROS

measurement.

Quantification of Tissue Damage

To assess the formation of visible lesions, wildtype, agb1-2, and gpa1-4 plants were

exposed to 500 or 700 ppb O3 for 3 hrs, then transferred to O3-free air. At 24 hours after

the onset of O3 exposure, plants were examined for visible lesions; plants with lesions

were photographed with a digital camera (Progres 3012, Kontron elektronik, Germany)

and the number of plants showing visible lesions was recorded. To measure tissue

damage by ion leakage, 18 leaves from 2 plants were collected at the time indicated in

each figure, rinsed with distilled water, then shaken in 25 ml distilled water on a rotary

shaker at 100 rpm for 4 hrs at room temperature. The conductivity of the wash solution

(μS/cm) was determined using a Corning 316 conductivity meter (Corning Inc. Big Flats,

NY). The total ion content was obtained by determining the conductivity of the same

41

leaf-containing solution after autoclaving. The relative ion leakage was obtained by

dividing the conductivity of solution before and after autoclaving and the values further

normalized by dividing them by the value obtained with a control sample of leaves from

plants not exposed to O3.

ROS Assays*

Frozen plant tissue was hand-ground with liquid nitrogen, the powder were weighed

and immediately taken up in 10 mM Tris-HCl buffer (pH 7.3). The extract was

centrifuged twice at 15000 rpm for 5min. ROS production was assayed at each time point

after the addition of 100 mM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) in

dimethyl sulfoxide (DMSO) to a final concentration of 10 μM. Fluorescence was

quantified using a VersaFluor fluorometer (Bio-Rad, Hercules, CA). Total protein was

quantified using a BioRad DC protein assay kit (Bio-Rad, Hercules, CA). The average

fluorescence values obtained from three successive measurements were divided by the

protein content and expressed as relative fluorescence units (RFU) per mg protein. These

values were then expressed as a ratio of RFU obtained with O3-exposed and control

plants.

RNA Preparation and Analysis

Leaf tissues were flash-frozen in liquid N2 immediately after removal from the

plants and stored at –80°C. Total RNA was isolated using RNEasy plant RNA isolation

42

*: ROS assay experiments in this part were done by Junghee Joo.

kit (Qiagen, Valencia, USA). Ten micrograms of total RNA from control and O3-treated

leaf tissue was fractionated on a 1.2% agarose/0.4 M formaldehyde RNA gel and

transferred to Hybond N+ nylon membrane (Amersham-Pharmacia, Buckinghamshire,

England). Membranes were stained with methylene blue to visualize the rRNA to confirm

equal loading. Probes were cloned from Arabidopsis cDNA made as described before

with primers GPA1 5’-ATGGGCTTACTCTGCAGTA-3' and 5'-CATAAAAGGC

CAGCCTCCAGT-3'; AGB1 5’-TCAAATCACTCTCCTGTGTCCTCC-3’ and

5’-TGTCTGTCTCCGAGCTCAAAGAACG-3’; and labeled using the ReadyPrime DNA

labeling kit (Amersham -Pharmacia, Buckinghamshire, England) with [α32P]dCTP (MP

Biomedicals Inc., Irvine, USA). Blots were hybridized and washed according to standard

procedures.

Protein Analysis

Total proteins were extracted from frozen tissues using a buffer containing 50 mM

Tris-HCl, pH 7.6, 1mM EDTA, 1% Triton, 1% SDS, 5mM DTT, 1 mM PMSF and

quantified using the BioRad’s DC Protein Assay kit, which is compatible with Triton and

SDS (Bio-Rad, Hercules, CA). Equal amounts of total membrane protein were loaded on a

12% polyacrylamide discontinuous gel (Biorad Mini electrophoresis system). After

electrophoresis, proteins were transferred to Hybond-P PVDF membrane (Amersham,

Piscataway, NJ) using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad, Hercules,

CA). Immunoblotting was performed with rabbit polyclonal anti-Gα antibody (kindly

43

provided by Dr. Alan Jones). After incubation with horseradish peroxidase-conjugated

anti-rabbit IgG antibodies, proteins were detected using ECL Plus Western Blotting

Detection Reagents (Amersham, Piscataway, NJ) according to the manufacture’s

instruction.

Results

Altered Ozone Sensitivity in G protein Mutant Plants

The absence of gene transcript for AtGPA1 and AtAGB1 was confirmed by reverse

transcriptase-mediated PCR in gpa1-4, agb1-2 and agb1-2/gpa1-4 double mutant plants,

as shown in Fig.1A. We exposed wildtype and mutant plants to either 500 ppb (parts per

billion) or 700 ppb (parts per billion) O3 for 3 hours and then checked plants for visible

lesions 24 hours after the onset of O3 exposure. Selected plants were photographed (Fig.

1B) and the number of plants showing visible lesions was counted (Fig. 1C). The gpa1-4

mutant plants showed less tissue damage than wildtype plants, while agb1-2 mutant and

double mutant plants showed more extensive tissue damage after either O3 treatment. To

quantify cell death after O3 treatment, we monitored ion leakage, an indicator of tissue

damage, immediately after O3 treatment in wildtype and mutant plants (Fig. 1D). Thus,

agb1-2 mutant and double mutant plants are more sensitive to O3 damage than wildtype

plants; while gpa1-4 mutant plants are less sensitive to O3 damage.

44

A.

B.

C. D.

gpa1-4 Col-0 agb1-2 gpa1-4/ agb1-2

Ubiquitin

AtAGB1

AtGPA1

gpa1-4 Col-0 agb1-2 gpa1-4/ agb1-2

500 ppb O3

700 ppb O3

0

20

40

60

80

Col-0 agb1-2 gpa1-4 agb/gpa

Lesi

on p

lant

s (%

)

500 ppb Ozone700 ppb Ozone

0

20

40

60

80

Col-0 agb1-2 gpa1-4 agb/gpa

Lesi

on p

lant

s (%

)

500 ppb Ozone700 ppb Ozone

Rel

ativ

e io

n le

akag

e

0

1

2

3

4

5

Col agb1-2 gpa1-4 agb/gpa

500 ppb Ozone700 ppb Ozone

Rel

ativ

e io

n le

akag

e

0

1

2

3

4

5

Col agb1-2 gpa1-4 agb/gpa

500 ppb Ozone700 ppb Ozone

Figure 1. Altered ozone sensitivity in G protein mutant plants A. Reverse transcriptase–mediated PCR analysis of the AtAGB1 and AtGPA1 transcript in wildtype (wt), gpa1-4, agb1-2 and gpa1-4/agb1-2 mutant plants. B. Photographs of plants with and without leaf damage (red circle) after indicted O3 treatment. C. The percentage of 4-week-old plants with visible lesions 24 h after a 3-h exposure to O3 at the indicated concentrations. D. Ion leakage (see Methods and Materials) was assayed 24 h after a 3-h exposure of 4-week-old plants to indicated O3 treatment (n=6).

45

Differential Expression Profile of GPA1 and AGB1 Gene after Ozone Treatment

Different functions of Gα and Gβ subunits in the oxidative stress response are

suggested by the different transcription profiles of AtGPA1 and AtAGB1 genes over the

course of the oxidative stress response to O3. Following O3 treatment for 6h at 350ppb,

we monitored the AtGPA1 and AtAGB1 transcript level by Northern blotting and the

AtGPA1 protein level by Western blotting. Blots were quantified using NIH Image

software. Transcript levels of both the AtGPA1 and AtAGB1 genes increased rapidly in

response to O3, peaking at about 1 hr after the onset of O3 exposure (Fig. 2A and 2B). The

AtGPA1 gene showed a second later increase in transcript level, which was not observed

for the AtAGB1 gene (Fig. 2A and 2B). Fig. 2C further showed that both the first and

second peaks of AtGPA1 transcript abundance were followed by increases in GPA1

protein levels, as detected by Western blotting with anti-GPA1 antibodies. Given the

opposite phenotypes of the gpa1-4 and agb1-2 mutant plants with respect to O3 damage,

the differential expression profile of AtGPA1 and AtAGB1 gene after ozone treatment

supported the concept that Gα and Gβ subunits play separable roles in the oxidative stress

response to O3.

46

Perturbed O3-Induced Oxidative Burst in G protein Mutants

The oxidative burst is believed to be an important signal mediating gene expression,

cell death and adaptive responses to protect plants from biotic and abiotic stress damage

(Lamb and Dixon, 1994; Levine et al., 1994; Lamb and Dixon, 1997; Baker and Orlandi,

Figure 2. Different expression profiles of

Gβ (B) transcript levels were

GPA1 and AGB1 genes after ozone treatment Gα (A) andmeasured by Northern blotting and blots were scanned and quantified using Image J. Gα protein (C) was detected by protein gel blotting with Gα antibodies; the blots were scanned, quantified using Image J and the values normalized by the amount present at the initial time point.

G-beta

0

10000

20000

30000

40000

50000

0 2 4 6 8 10 12

Time (h)

Int.

Den

sity

ControlOzone

G-alpha

0

10000

20000

30000

40000

50000

0 2 4 6 8 10 12

Time (h)In

t. D

ensi

ty

Controlozone

A. B.

G-alpha protein

0

0.4

0.8

1.2

6

0 3 6 9 12 15 18 21 24

Time (h)

Rel

ativ

e In

tens

it

C. 1.

y

controlozone

47

1999; Droge, 2002; Inze and Van Montagu, 2002). We measured the oxidative burst,

predominantly H2O2, as catalase-inhibitable fluorescence using the dye

2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) as described in Materials and

Methods. A 6-hr exposure of wildtype plants to 350 ppb O3 evokes a biphasic oxidative

burst with a first peak at about 1 hour of exposure, followed by a second possibly

bimodal peak between 12 and 24 hrs after the onset of exposure (Fig. 3A). The first peak

is markedly reduced and the late peak is totally absent in O3-exposed gpa1-4 mutant

plants (Fig. 3B). By contrast, while the first peak is missing in ozone-exposed agb1-2

mutant plants (Fig. 3C), the second peak is similar to that in wildtype plants. These

observations imply that the early peak requires the intact heterotrimeric G protein or both

Gα and Gβ subunits, whereas the late peak requires Gα subunit but not Gβ subunit or

formation of the heterotrimer.

A. Col-0 B. gpa1-4

0

1

2

3

0 3 6 9 12 15 18 21 24Time (hr)

RFU

4

- ozone+ ozoneO3

control

O30

1

2

3

0 3 6 9 12 15 18 21 24Time (hr)

RFU

4-ozone+ ozoneO3

control

O3

4 4

0

1

2

3

0 3 6 9 12 15 18 21 24Time (hr)

RFU

- ozone+ ozoneO3

control

O3O30

1

2

3

0 3 6 9 12 15 18 21 24Time (hr)

RFU

-ozone+ ozoneO3

control

O3O3

48

C. agb1-2

were done by Junghee Joo.

Enhanced Resistance to O

Figure 3. Perturbed O3-induced oxidative burst in G protein mutants*

pa1-4 (B), and gb1-2 (C) plants were exposed to 350 ppb

0

1

2

3

4

0 3 6 9 12 15 18 21 24Time (hr)

RFU

- ozone

*: ROS assay experiments in this part

3 Damage by Lower Dose O3 Pretreatment

of A. thaliana to

infe

It has been observed that ozone exposure induces resistance

ction with virulent phytopathogenic Pseudomonas syringae strains (Sharma et al.,

1996). This phenomenon is referred to as “cross protection” or “cross tolerance”. To

determine if G protein signaling is attributed to enhanced O3 tolerance by cross protection

with lower dose O3 pretreatment, we briefly exposed plants to 350 ppb O3 (2 hours) and

let them recover in the air for 2 hrs. We then exposed plants to 700 ppb O3, which causes

tissue damage, to examine whether they showed a higher tolerance to O3. As shown in

Fig. 3D, the pretreatment rendered wildtype plants, agb1-2 and double mutant plants less

sensitive to tissue damage compared with plants without pretreatment, although gpa1-4

mutant plants exhibited little tissue damage with or without pretreatment because of their

marked enhanced O3 resistance (Figure 4). Thus, pretreatment with lower dose of O3

+ ozoneO3

control

O3

0

1

2

3

4

0 3 6 9 12 15 18 21 24Time (hr)

RFU

- ozone (A) to (C) Wild-type (A), g

control+ ozoneO3

aO3 for 6 h (closed circles) or air (open circles), then maintained in O3-free air; leaves were collected and frozen at the indicated times. ROS were measured by H2DCFDA fluorescence; values were normalized by the total protein concentration in leaf extracts, and relative fluorescence units (RFU) were calculated as a ratio of the value obtained at the indicated time to the control (n=5).

O3O3

49

increased their ability to withstand higher dose of O3, which suggested that biochemical

or molecular changes that occured within the first few hours after 350 ppb O3 exposure

play a role in eliciting protective responses from cell death. However, the agb1-2 and

double mutant plants remained more sensitive to O3 damage than wildtype plants even

with pre-treatment, which suggested that Gβ protein or the Gβγ complex contributes to

the rapid enhancement of ozone tolerance observed following O3 pretreatment. Given that

agb1-2 and double mutant plants still showed some enhancement of O3 tolerance after

pretreatment, Gβ protein or the Gβγ complex was not solely responsible for the

development of O3 tolerance.

0

1

2

3

4

5

Rel

ativ

e Io

n le

akag

O3 pretreatment No pretreatment

e

Col-0 gpa1-4 agb1-2 agb1-2/gpa1-4

Figure 4. Enhanced resistance to O3 d mage by lower does O3 pretreatment Mutant and wild-type plants (4 weeks old) were exposed to O3 350ppb for 2 h, transferred

hich leaves

a

to O3-free air for 2 h, and then exposed to 700 ppb ozone for 3 h, following wwere collected and assayed for ion leakage as described in Methods and Materials (n=6).

50

Discussion

The results show that Gα and Gβ subunits of the Arabidopsis heterotrimeric G protein

are

te Arabidopsis heterotrimeric G protein either

dire

required for development of the first component of the oxidative burst. On the other

hand, the absence of the early ROS peak, which is present in O3-exposed wildtype plants,

in the gpa1-4 and the agb1-2 mutant plants indicates that ROS derived from O3 and

dissolved in the apoplast is not the source of ROS burst and it can not activate

endogenous ROS production without the heterotrimeric G protein. Thus, exposure to

external ROS requires G protein to transduce or amplify signals to further activate the

endogenous ROS-generating system. This raises an intriguing question about how the

heterotrimeric G protein is activated by extracellular ROS and how it activates the

intracellular ROS-generating systems.

The extracellular ROS can activa

ctly or indirectly. Two mammalian Gα proteins, Gαi and Gαo, are ROS targets and

directly regulated by H2O2 (Nishida et al., 2000; Nishida et al., 2002). H2O2 treatment

increases the GTP affinity of the Gα subunit in a dose-dependent manner and activates

Gβγ signaling and its downstream effectors (Nishida et al., 2000). Cys287 in Gαi and Gαo

(Nishida et al., 2002), which is required for their ROS activation, is conserved in the

AtGα1 protein (Ma et al., 1990). Thus it is possible that the Arabidopsis G protein itself is

redox regulated. However, we can not exclude the possibility of the indirect activation of

51

G protein by the extracellular ROS. It could be a G protein receptor or another interacting

protein that is oxidized to activate G protein signaling for the ROS-generating system.

A small GTPase has been implicated in the downstream of the heterotrimeric G

protein in the activation of membrane bound NADPH oxidase in the pathogen response

of rice (Suharsono et al., 2002). Phospholipase Dα (PLDα), which hydrolyzes

phospholipids into free head groups and phosphatidic acid (PA), is another potential

intermediate molecule for ROS production mediated by G protein. Phosphatidic acid (PA)

as a lipid messenger has been shown to promote the production of superoxide, which is

decreased in the PLDα mutant plant (Sang et al., 2001). Moreover, the direct effect of Gα

on the activity of PLDα has been demonstrated by their physical interaction (Zhao and

Wang, 2004). Since no downstream targets of Gβγ signaling have yet been identified in

plants, it is hard to rationalize the role of Gβγ in activating the component of ROS burst.

In addition to their role in ROS production in the oxidative stress response, Gα and

Gβ subunits of the heterotrimeric G protein may serve different functions in cell death

associated oxidative stress response to O3. The observation that the late peak of ROS

occurs at 20 h after the onset of O3 exposure and is coincident with cell death as well as

the development of large lesions in plants, allows us to hypothesize that the late peak

mediated by Gα/NADPH-oxidase mainly contributes to cell death.

Although the resistance of gpa1-4 mutant plants to O3 damage may be attributable

to the absence of the late ROS peak, what causes the hypersensitive phenotype of the

agb1-2 mutant and double mutant to O3 damages is still unclear. Nonetheless, the

52

phenotype of the double mutant is similar to that of the agb1-2 single mutant, suggesting

that Gβ act predominantly in cell death associated oxidative stress response to O3. The

opposite phenotypes of the gpa1-4 and agb1-2 mutants have been seen in other biological

process. For example, agb1-1 mutant plants have increased apical dominance whereas

gpa1-1 mutant plants have decreased apical dominance. Moreover, agb1-1 and agb1-2

have more root mass than wild-type, whereas gpa1-1 and gpa1-2 have less root mass than

wild-type (Ullah et al., 2003). However, similar phenotypes of the gpa1-4 and agb1-2

mutants at various developmental stages are also found. For example, fruit and seed

weights are greater for both mutants than wildtype plants. Since we know little about the

action of heterotrimeric G protein in Arabidopsis, it is hard to predict how Gα and Gβγ

subunits play common and separable roles in their heterotrimeric complex or what causes

them dissociate from complex to interact with their downstream effectors.

53

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Droge, W. (2002). Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47-95.

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Joo, J.H., Wang, S., Chen, J.G., Jones, A.M., and Fedoroff, N.V. (2005). Different signaling and cell death roles of heterotrimeric G protein alpha and beta subunits in the Arabidopsis oxidative stress response to ozone. The Plant Cell 17, 957-970.

Kwak, J.M., Mori, I.C., Pei, Z.M., Leonhardt, N., Torres, M.A., Dangl, J.L., Bloom, R.E., Bodde, S., Jones, J.D., and Schroeder, J.I. (2003). NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. Embo J 22, 2623-2633.

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Levine, A., Tenhaken, R., Dixon, R., and Lamb, C. (1994). H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79, 583-593.

Ma, H., Yanofsky, M.F., and Meyerowitz, E.M. (1990). Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana. Proc Natl Acad Sci U S A 87, 3821-3825.

Mahalingam, R., Gomez-Buitrago, A., Eckardt, N., Shah, N., Guevara-Garcia, A., Day, P., Raina, R., and Fedoroff, N.V. (2003). Characterizing the stress/defense transcriptome of Arabidopsis. Genome Biology 4, R20.

Mason, M.G., and Botella, J.R. (2000). Completing the heterotrimer: isolation and characterization of an Arabidopsis thaliana G protein gamma-subunit cDNA. Proc Natl Acad Sci U S A 97, 14784-14788.

Mason, M.G., and Botella, J.R. (2001). Isolation of a novel G-protein gamma-subunit from Arabidopsis thaliana and its interaction with Gbeta. Biochim Biophys Acta

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alpha(i) and G alpha(o) are target proteins of reactive oxygen species. Nature 408, 492-495.

Nishida, M., Schey, K.L., Takagahara, S., Kontani, K., Katada, T., Urano, Y., Nagano, T., Nagao, T., and Kurose, H. (2002). Activation mechanism of Gi and Go by reactive oxygen species. J Biol Chem 277, 9036-9042.

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56

Chapter 3

BOTH HETEROTRIMERIC G PROTEIN SIGNALING AND ROSIGNALING

ARE IMPLICATED IN THE ARABIDOPSIS UNFOLDED PROTEIN RESPONSE

Abstract

We present evidence that heterotrimeric G protein signaling and ROS signaling are

involved in UPR-associated cell death in Arabidopsis. Seedlings of agb1-2 mutant plants

with a null mutation in the gene coding for the β subunit of the heterotrimeric G protein

are more resistant to growth inhibition by the protein glycosylation inhibitor tunicamycin

(Tm) than wildtype plants and gpa1-4 plants with a null mutation in the gene encoding

the α subunit of the heterotrimeric G protein. Leaves of agb1-2 mutant plants exhibit

markedly less cell death after Tm treatment than those of wildtype plants. The

transcriptional response of agb1-2 mutant plants to Tm is less pronounced than that of

wildtype plants. On the other hand, AtrbohD (AtrbohD null mutation) and AtrbohD/F

(AtrbohD and AtrbohF null mutation) mutant plants show more cell death in response to

treatment with Tm than wildtype plants and AtrbohF (AtrbohF null mutation) mutant

plants. The protective role of the endogenous ROS elicited by Tm treatment against

Tm-induced cell death is also seen using ROS scavengers. Moreover, the transcriptional

response is delayed by the pretreatment with ROS scavengers.

A majority of the Arabidopsis Gβ protein is associated with the endoplasmic

57

reticulum (ER) and is degraded after Tm treatment, while Gα protein is not. Collectively,

these observations indicate that the Gβγ complex, not Gα, plays an important role in cell

death associated with the unfolded protein response in Arabidopsis. Moreover these

observations imply a direct role for both ROS and G protein signaling in the UPR.

Introduction

The endoplasmic reticulum (ER) is the organelle in which secretory proteins are

synthesized, modified and delivered to their target sites. ER stress occurs when protein

folding, modification and transportation are perturbed. Cells from ER stress activate a

number of biochemical changes termed the unfolded protein response (UPR) (Schroder

and Kaufman, 2005; Wu and Kaufman, 2006).

In mammalian cells, the three distinct protective responses of the UPR are

believed to be activated by ER-resident IRE1 (inositol-requiring transmembrane kinase

and endonuclease 1), ATF6 (activation of transcription factor 6) and PERK (protein

kinase–like ER kinase) respectively (Schroder and Kaufman, 2005). These three

proteins are bound to the ER-resident luminal binding protein (BiP) under resting

conditions. When the ER is stressed, BiP preferentially binds to the unfolded proteins,

effectively being titrated away from IRE1, ATF6 and PERK. The dissociation from BiP

activates PERK, which phosphorylates the translation elongation factor eIF2α and

furthermore inhibits translation of most new proteins (Bertolotti et al., 2000; Liu et al.,

58

2000). After dissociation from BiP, ATF6 is translocated to the Golgi and cleaved by

S1/S2 protease to form active ATF6, which is a transcription factor promoting

chaperone gene expression (Li et al., 2000; Wang et al., 2000). Upon its release from

BiP, IRE is activated by dimerization and auto-phosphorylation. The active IRE cleaves

the precursor of X-box binding protein from XBP1u mRNA to mature XBP1s mRNA,

which is then translated into a transcription factor to induce the expression of genes

involved in degradation of unfolded proteins (Yoshida et al., 2001). In addition to these

pathways, the UPR also enhance ER-associated protein degradation (ERAD) to remove

unfolded or misfolded proteins from their target sites (Kruse et al., 2006). Perturbation

of any of these processes in the UPR can cause cells or organisms to be susceptible to

ER stress and cell death, which is involved in certain diseases such as diabetes,

Alzheimer’s, atherosclerosis, and cancer (Wu and Kaufman, 2006).

Although the study of the UPR and ER stress in plants is not as extensive as that in

animals, it has been shown that plants have a protective transcriptional response when

exposed to ER stress, including induction of genes encoding protein-folding enzymes and

chaperones (Martinez and Chrispeels, 2003; Kamauchi et al., 2005). Both tunicamycin

(Tm), an inhibitor of N-linked protein glycosylation and cyclopiazonic acid (CPA), an

inhibitor of the ER-type II Ca2+ pump, can trigger the UPR and programmed cell death in

cultured plant cells (Zuppini et al., 2004). However, only a few orthologs of mammalian

UPR proteins have been identified so far in plants. These are Ire1 (Noh et al., 2002),

eIF2α (Chang et al., 1999; Chang et al., 2000) and p58IPK (Bilgin et al., 2003) . The

59

Arabidopsis genome has no orthologs of the ER stress-induced XBP1 or ATF6 genes,

although other transcription factors like bZIP60 (Iwata and Koizumi, 2005) and NTM1

(Kim et al., 2006) contain a trans-membrane domain, which can be cleaved by a

peptidase and hence may function in similar manner as ATF6.

Plants respond to both biotic and abiotic stress by producing ROS (reactive oxygen

species), which are believed to be a fundamental part of intracellular signal transduction

and intercellular cell communication (Fedoroff, 2006). The redox regulation of the

transcriptional regulatory factor NPR1, which promotes UPR gene expression in systemic

acquired resistance (SAR), appears to suggest an interconnection between ROS

homeostasis and UPR induction in plants (Mou et al., 2003; Wang et al., 2005). Moreover,

both ROS signaling and UPR have been demonstrated to be a critical part of the plant’s

defense response (Fedoroff, 2006). However, the functional interaction between them has

not yet been investigated.

We have reported that the Gα and Gβ subunits serve both separable and synergistic

functions in signaling by reactive oxygen species (ROS) in the oxidative stress response

to O3 (Joo et al., 2005). The Arabidopsis genome encodes a single canonical

heterotrimeric G protein and one α subunit (Ma et al., 1990), one β subunit (Weiss et al.,

1994), and two γ subunits (Mason and Botella, 2000, 2001). Here we show that signaling

through the Gβ subunit of heterotrimeric G protein plays a direct role in ER-stress

associated cell death and the biphasic oxidative burst induced by Tm exerts a protective

effect against ER-stress associated cell death. We show that plants homozygous for a null

60

mutation of the Gβ subunit of heterotrimeric G protein are substantially more resistant to

Tm-induced cell death than either wildtype plants or plants homozygous for a null

mutation of the Gα subunit. We also show that the transcriptional response of the agb1-2

mutant plants (Gβ null mutation) is reduced and delayed compared with that of wildtype

plants and that the large protein aggregates characteristic of the ER stress are not

observed in mutant plants. We show that interference with ROS production by a flavin

oxidase inhibitor, a ROS scavenger or mutations that affect the AtrbohD and AtrbohF

genes increases the sensitivity of Arabidopsis plants to Tm-induced cell death and delays

the expression of UPR chaperones and other markers. A majority of the Gβ protein is

detected in the ER, judged by ER marker protein BiP. Moreover, the Gβ protein is

degraded during the UPR. These observations strongly suggest the Gβ subunit of the

heterotrimeric G protein plays a direct role in cell death signaling in the UPR.

Methods and Materials

Plant Materials, Growth Conditions, and Tm Treatment

We used Arabidopsis thaliana Col-0 plants, homozygous agb1-1 (Lease et al.,

2001), agb1-2, gpa1-4, and agb1-2/gpa1-4 double mutants (Ullah et al., 2003), all

characterized as transcript-null mutants in the Col-0 background and atrbohD, atrbohF,

and atrbohD/F double mutants in the Col-0 background (Torres et al., 2002). Plants were

grown in MetroMix 200 (Scotts-Sierra Horticultural Products Company, Marysville, OH)

61

in 5-cm pots at 65% humidity under fluorescent light at 30 W/m2/s with 14-h light/10-h

dark photoperiod for 5 weeks. Five rosette leaves of similar size were infiltrated

sub-epidermally on lower surface with 15-30 μg/ml Tm (Sigma-Aldrich, St. Louis, MO)

in 1.6% DMSO using a needleless syringe. Controls were infiltrated with the same

DMSO solution lacking Tm. Where indicated, plants were pre-treated with 20 μM

diphenyleneiodonium sulfate (DPI) (Sigma-Aldrich), a flavin oxidase inhibitor, or 2 mM

N-acetylcysteine (NAC) (Sigma-Aldrich), reactive oxygen species (ROS) scavenger, for

4 hours prior to Tm injection either by watering.

Quantification of Leaf Senescence and Cell Death.

Chlorophyll degradation, an indicator of leaf senescence, was measured 8 days after

Tm injection as described (Arnon, 1949). The chlorophyll measurement was normalized

to the wet leaf weight. Release of electrolytes was used to quantify cell damage and death.

To measure electrolyte release, 5 injected leaves from a single plant were collected each

day after inoculation and shaken in 15 ml distilled water at 100 rpm for 4 h at room

temperature. The conductivity of the wash solution (mS/cm) was determined using a

portable conductivity meter (Control Company, Friendswood, TX). The total electrolyte

content was obtained by measuring the same leaf-containing solution after autoclaving.

Six replicates were averaged to calculate the final fraction of electrolytes released as a

fraction of the total electrolyte content. Relative electrolyte leakage is the ratio of the

fraction obtained with leaves of treated plants to that obtained with leaves from untreated

62

control plants. Cell death was detected histochemically by trypan blue staining as

described (Pasqualini et al., 2003).

RNA Isolation and Northern Blot Hybridization

Leaves were fresh frozen in liquid N2. Total RNA was isolated using Trizol reagent

(Invitrogen, Carlsbad, CA). Ten micrograms of total RNA from control and Tm-injected

leaf tissue were fractionated on a 1.2% agarose/0.4 M formaldehyde gel and transferred

to Hybond N+ nylon membrane (Amersham-Pharmacia, Piscataway, NJ). Membranes

were stained with methylene blue to visualize rRNA as loading controls. Probes were

cloned from Arabidopsis thaliana Col-0 cDNA, which was made as previously described

with the following gene-specific primers Bip3: 5-ATGATTTTTATCAAGGAAAACACA

GCG-3 and 5-TTACCAAGGGCTTTGTGATCC; Bip1&2: 5-GGCTCGCTCGTTTGGA

GCAAA-3 and 5-CCGTTATCAAT GGTCAAGACACT-3; PDI: 5-ATGGCGAAATCTC

AGATCTGGTT-3 and 5-GTCCCTGCTGGTCCCAGATTT-3. The probes were labeled

using the ReadyPrime DNA labeling kit (Amersham-Pharmacia) with [α32P]dCTP (MP

Biomedicals Inc., Irvine, CA). Blots were hybridized and washed according to

manufacture’s instructions (Sigma-Aldrich).

ROS Assays

Frozen plant tissues were hand-ground with liquid nitrogen immediately after Tm

treatment, the powder were weighed and immediately taken up in 10 mM Trs-HCl buffer

63

(pH 7.2). The extract was centrifuged twice at 15,000 rpm for 5min. ROS production was

assayed at each time point after the addition of 100 mM 2',7'-dichlorodihydrofluorescein

diacetate (H2DCFDA) in dimethyl sulfoxide (DMSO) to a final concentration of 10 μM.

Fluorescence was quantified using a VersaFluor fluorometer (Bio-Rad, Hercules, CA).

Total protein was quantified using a BioRad DC protein assay kit (Bio-Rad, Hercules,

CA). The average fluorescence values obtained from three successive measurements were

divided by the protein content and expressed as relative fluorescence units (RFU) per mg

protein. These values were then expressed as a ratio of RFU obtained with Tm infiltrated

and control plants.

Cellular Membrane Fraction

Five week-old transgenic plants containing a CFP-Gβ fusion protein expressed from

the AGB1 promoter were homogenized using buffer containing 50 mM Tris-MES, pH 7.5,

1 mM dithiothreitol, 5 mM EDTA, 0.3 M sucrose and protease inhibitors. The

homogenate was filtered through a double layer of Miracloth (EMD Biosciences, Inc. La

Jolla. CA) and centrifuged at 3,200xg for 10 min (JA 20, Beckman Coulter, Inc. Fullerton

CA). The supernatant was further pelleted by centrifugation at 100,000xg (TY70 Ti,

Beckman) to obtain microsomal membranes, which were then subjected to aqueous

two-phase partitionining (Chen et al., 2002; Kato et al., 2004). The microsomal pellet was

suspended in a solution containing 0.3 M sucrose, 3 mM KCl, and 5 mM KH2PO4 and

adjusted to 6.4% (w/w) each of Dextran T500 and PEG3350. The phases were separated

64

by centrifugation at 750xg (Thermo IEC 243) and the phase partitioning was repeated 3

times. The upper and lower phases were diluted separately with buffer containing 0.3 M

sucrose, 3 mM KCl, 1 mM EDTA and centrifuged at 100,000g (TY70 Ti, Beckman) to

obtain plasma membrane (Pm) and intracellular membrane (Im) fractions. The Pm

fraction was dissolved in SDS-PAGE sample buffer for analysis and Im fraction was

re-suspended in buffer containing 25 mM Tris (pH 7.5), 10% sucrose, 1 mM dithiothreitol,

2 mM EDTA, and protease inhibitors and sedimented through 10-50% sucrose density

gradient at 100,000xg for 16 h (SW28 3707, Beckman). Fractions (0.8 ml) were collected,

concentrated using StrataClean Resin (Stratagene, Cedar Creek, TX) and dissolved in

SDS-PAGE sample buffer for analysis. Isolated membrane fractions were loaded on a

10% polyacrylamide discontinuous gel (Bio-Radmini Electrophoresis System). After

electrophoresis, proteins were transferred to Hybond-P PVDF membrane (Amersham,

Piscataway, NJ) using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad).

Immunoblotting was performed as described below.

Protein Analysis

Total proteins were extracted using extraction buffer (50mM Tris-HCl, pH 7.6, 1

mM EDTA, 1% Triton, 2% SDS, 5 mM DTT, and 1 mM PMSF) and quantified using

Bio-Rad’s DC protein assay kit, which was compatible with Triton and SDS. Equal

amounts of total membrane protein were loaded on a 12% polyacrylamide discontinuous

gel (Bio-Radmini electrophoresis system). After electrophoresis, proteins were

65

transferred to Hybond-P PVDF membrane (Amersham, Piscataway, NJ) using a Mini

Trans-Blot electrophoretic transfer cell (Bio-Rad). Immunoblotting was performed with

goat polyclonal anti-BiP antibodies (1:250, Santa Cruz Biotechnology, Inc., Santa Cruz,

CA) and horseradish peroxidase–conjugated anti-goat IgG antibodies (Santa Cruz

Biotechnology) for BiP protein analysis. The immunoblot was incubated with mouse

monoclonal anti-CFP antibodies (1:2000, BD Bioscience, Mountain View, CA) and

horseradish peroxidase–conjugated anti-mouse IgG antibodies for AtAGB1 protein level

measurement. The above blot was striped and probed with rabbit polyclonal anti-Gα

antibodies and horseradish peroxidase–conjugated anti-rabbit IgG antibodies (Invitrogen).

Proteins were detected with ECL Plus protein gel blotting detection reagent (Amersham)

according to the manufacturer’s instructions.

Results

Plants Lacking the Heterotrimeric Gβ Subunit are Tm-Resistant.

To compare the Tm sensitivity of G-protein mutants with Arabidopsis wildtype plants,

seeds of plants homozygous for null mutations in the genes encoding the Gα and Gβ

subunits, as well as doubly mutant plants, were plated on Tm-containing medium for 6

days, then transferred to tunicamycin-free agar medium for 10 days. Tm inhibited the

growth in gpa1-4 (Gα null mutation) seedlings and wildtype seedlings, while

homozygous agb1-2 (Gβ null mutation) seedlings and doubly mutant gpa1-4/agb1-2

66

seedlings were much less sensitive than either wildtype or gpa1-4 seedlings (Fig. 5).

To observe the effect of Tm on adult plants, rosette leaves of 5 weeks plants were

infiltrated sub-epidermally on the lower surface by Tm. Wildtype plants infiltrated with

Tm showed large areas of leaf senescence and cell death by 6 days after injection (Fig.

6A), whereas, leaves of agb1-2 plants showed small areas of damage (Fig. 6A).

Close-up views also showed larger areas of tissue collapse and cell death in injected

wildtype leaves than agb1-2 mutant leaves, which have restricted damaged areas around

the injection site (Fig. 6B). Vital dye staining with Trypan blue also revealed much larger

areas of dead cells in wildtype than in agb1-2 mutant leaves (Fig. 6B). Similar results

were obtained with leaves of plants homozygous for a second mutant allele, agb1-1, of

the gene coding for the Gβ subunit of the heterotrimeric G protein (data not shown).

Col-0

gpa1-4

agb1-2 gpa1-4/ agb1-2

Control Tm

Figure 5. Plants homozygous for null mutations in genes coding for the α and β subunits of the heterotrimeric G protein exhibited different sensitivities to Tm induced growth inhibition. Homozygous gpa1-4 (Gα null mutation), agb1-2 (Gβ null mutation), and gpa1-4/agb1-2 double mutants were germinated on agar medium containing 0.3 μg/ml Tm for 6 days, then transferred to medium without Tm, allowed to recover for 10 days, and photographed.

67

A. Control Tm

Col-0 agb1-2 Col-0 agb1-2

B.

Thus, mutant plants lacking the Gβ

Col-0 agb1-2

Control Tm Control Tm

Figure 6. Leaf senescence and cell death in wildtype and agb1-2 mutant plants A 15 g/ml Tm solution in 1.6% DMSO was infiltrated into leaves as described in Methods. Plants were photographed at 6 days after infiltration (A) and leaves were photographed at 8 days (B, left panel) or cleared and stained with trypan blue (B, right pane s were harvested for daily to determine electrolyte release as described (C); f nt at 8 days (D)

μ

l); leave

or determination of chlorophyll conte0

5

10

15

20

25

30

0 1 2 3 4 5 6 7Time (days)

% E

lect

roly

te re

leas

e

WTagb1-2

C.

ConTm

150

190

230

270

310

Chl

orop

hyll

(mg/

g

D.

)

agb1-2 Col-0

68

Cell damage was also quantified by measuring the release of intracellular electrolytes

to the extracellular space and leaf senescence was measured by loss of chlorophyll.

Wildtype leaves showed a transient increase in the electrolyte release a day after Tm

treatment, recovered transiently, then showed a progressive increase in the release of

intracellular electrolytes as the area of leaf damages enlarges (Fig. 6C), while

homozygous agb1-2 mutant plants only exhibited a little enhanced ion leakage after Tm

treatment. Similarly, wildtype leaves exhibited much more extensive loss of chlorophyll

than leaves of abg1-2 mutant plants (Fig. 6D). Thus, plants lacking β subunit of the

heterotrimeric G protein are more resistant to Tm-induced cell death than wildtype plants,

suggesting that Gβ subunit palys a role in cell death response induced by ER stress.

The Transcriptional and Translational Response to Tm is Muted in Gβ Mutants

The induction of several characteristic ER stress markers occurred in wildtype and

agb1-2 mutant plants after Tm treatment. The abundance of BiP transcripts, particularly

for BiP3, increased rapidly, peaking at about 3 hrs after Tm infiltration. The abundance of

PDI transcripts also increased, but peaks later at 6 hrs after Tm infiltration (Fig. 7A).

Both BiP and PDI mRNA levels declined at 9 hrs and then increased again at 12 hrs.

Although BiP and PDI transcript levels increased in agb1-2 mutant plants after Tm

infiltration, they increased more slowly than in wildtype plants and did not reach the

levels observed in wildtype plants. Moreover, BiP proteins did not accumulate to the

same levels in the agb1-2 mutants as in wildtype plants (Fig. 7B). Large aggregates

69

migrating more slowly than the BiP monomers can be detected in extracts from wildtype

plants, but are much less prominent in agb1-2 mutant plants. Thus the transcriptional and

translational responses to Tm-induced ER stress were much less marked in agb1-2 mutant

plants than in wildtype plants.

A.

agb1-2Col-0

BiP1&2

BiP3

PDI

control tunicamycin1 3 6 9 12 1 3 6 9 12 1 3 6 9 12 1 3 6 9 12

control tunicamycin

agb1-2Col-0

BiP1&2

BiP3

PDI

control tunicamycin1 3 6 9 12 1 3 6 9 12 1 3 6 9 12 1 3 6 9 12

control tunicamycin hours

B.

78KD

0 1 2 4 0 1 2 4 daysTm:

Col-0 agb1-2

48KD

78KD

0 1 2 4 0 1 2 4 daysTm:

Col-0 agb1-2

48KD

78KD

0 1 2 4 0 1 2 4 daysTm:

Col-0 agb1-2

48KD

Figure 7. The Tm-induced UPR is attentuated in agb1-2 mutant plants. A. Northern blot analyses of Col-0 and agb1-2 mutant plants treated with 15 μg/ml Tm. Total RNA was extracted at the indicated times and 10 μg total RNA was loaded into each lane. UPR marker probes are indicated on the right. 25s rRNA was used as internal loading control. B. Western blot analysis of Col-0 and agb1-2 mutant plants treated as in A. Total protein was extracted at the indicated times and 30 μg of protein was loaded into each lane. Anti-AtBiP1&2 was used to probe the membrane.

70

Tm Induces an Oxidative Burst, Perturbation of which Delays Expression of UPR Marker Genes

The production of ROS is induced by a variety of biotic and abiotic stressors. To

determine whether Tm-induced ER stress can trigger ROS production, we measured H2O2

level after Tm treatment. Our results showed that Tm induced a biphasic oxidative burst

in Arabidopsis leaves (Fig. 8A). The Tm treatment promoted a rapid transient increase in

H2O2 level which subsided by 3 hours, then increased again at 6 hrs and declined at 9 hrs

after the onset of treatment.

To further evaluate the role of ROS in UPR-associated gene expression, we inhibited

ROS production with the flavin oxidase inhibitor diphenylene iodonium (DPI) by

pretreatment before Tm application and examined the effect of the inhibitor on gene

expression in the UPR. Our results showed that inhibition of ROS production delayed and

attenuated the increase in the expression level UPR marker genes (Fig. 8B). Early

induction of BiP1&2, BiP3 and PDI mRNA levels at 3 hrs after the Tm treatment was

delayed until 6 hrs after Tm treatment in plants treated with the inhibitor. These

observations indicated that ROS are required for the activation of UPR-associated gene

expression in response to Tm.

71

A.

0.4

0.8

1.2

1.6

2

1 3 6 9Tm (h)

RFU

wt controlwt Tm

B.

1 3 6 9 12 1 3 6 9 12 1 3 6 9 12 1 3 6 9 12 hrs

control

BiP3

PDI

25S rRNA

BiP1+2

DPI Tm DPI+Tm

1 3 6 9 12 1 3 6 9 12 1 3 6 9 12 1 3 6 9 12 hrs

control

BiP3

PDI

25S rRNA

BiP1+2

DPI Tm DPI+Tm

Figure 8. Tm induces an oxidative burst and DPI delays UPR marker gene expression A. ROS production was measured by H2DCFDA fluorescence in plants treated with 15 μg/ml tunicamycin (Tm). Fluorescence values were normalized to the protein content of the leaf extracts and expressed as a ratio (RFU) of the values obtained at the indicated times to the control value at 0 time. B. Northern analysis of UPR marker genes in response to Tm treatment with and wiouout DPI pre-treatment. Wildtype (Col-O) plants were treated with buffer alone (control), or buffer containing 20 μM diphenylene iodonium (DPI), 15 μg/ml Tm (Tm); or both (DPI+Tm) 4 hrs. After pre-treatment, leaves of plants were infiltrated with Tm (15 μg/ml).

Endogenous ROS Protect against Tm-Induced Tissue Damage and Cell Death.

Endogenous ROS serve as both intra- and inter-cellular signals, as well as triggering

cell death (Torres et al., 2002; Joo et al., 2005). To assess the role of ROS in the

72

UPR-associated cell death, we pre-treated plants with diphenylene iodonium (DPI), a

flavin oxidase inhibitor, or N-acetyl cysteine (NAC), a ROS scavenger, for four hours

before Tm injection and observed the phenotype after 3 days. We found the area of cell

death around the site of Tm infiltration was substantially greater in leaves from plants

pre-treated with either DPI or NAC (Fig. 9A). We quantified Tm-induced cell death by

measuring electrolyte release. Electrolyte release from wildtype plants pretreated with

either DPI or NAC was roughly four-fold greater after Tm treatment than from plants

pretreated with the same buffer alone (Fig. 9B). Thus either inhibiting endogenous ROS

production or suppressing ROS exacerbated Tm-induced cell death in wildtype plants,

indicating that ROS serve a protective role in the UPR-associated cell death.

A. B.

Tm +DPI1

2

3

4

Tm NAC DPI Tm +NAC

Rel

ativ

e io

n le

akag

e

Tm +DPI1

2

3

4

Tm NAC DPI Tm +NAC

Rel

ativ

e io

n le

akag

e

Tm + DPI Tm + NAC Tm

DPI NAC Buffer

Tm + DPI Tm + NAC Tm

DPI NAC Buffer

Figure 9. Suppressing ROS production increases Tm-induced cell damage and death A. 5 week old plants were pre-treated either with buffer alone or buffer containing 20 μM DPI or 2 mM NAC, then leaves were injected with 15 μg/ml Tm. Control plants were pretreated with either buffer alone or with buffer containing either NAC or DPI, but not injected with Tm. After 3 days of treatment, photographs were taken. B. Electrolyte leakage was measured from leaves treated as above. The values reported are the ratio of the fraction of total ion content released by treated and control plants.

73

We have previously reported that both AtrbohD and AtrbohF contribute to cell death

signaling mediated by ROS between cells in response to oxidative stress (Torres et al.,

2002; Joo et al., 2005). To determine the role of ROS generated by AtrbohD and AtrbohF

oxidase in the UPR-associated cell death, plants homozygous for the atrbohD mutation,

the atrbohF mutation or doubly mutant atrbohD/F plants were infiltrated by Tm and cell

death induced by Tm was quantified by electrolyte release. The atrbohD plants and

doubly mutant atrbohD/F plants show much larger areas of cell death around the Tm

injection site than wildtype plants and atrbohF single mutants (Fig. 10A). Electrolyte

release from atrbohD mutant and doubly mutant atrbohD/F plants was only slightly

higher than that from wildtype plants and atrbohF single mutants 3 days after Tm

treatment. It then increased to a value about two-fold greater in atrbohD mutant and

doubly mutant atrbohD/F plants than wildtype plants and atrbohF single mutants 7 days

after Tm treatment (Fig. 10B). The susceptible phenotype of atrbohD mutant and doubly

mutant atrbohD/F plants to Tm induced cell death indicated that intercellular ROS

signaling prevents the over proliferation of UPR-associated cell death. Moreover, the

major contributor to such signaling is the AtrbohD NADPH oxidase, not AtrbohF

NADPH oxidase. Thus, both inhibitor studies and genetic evidence from mutants showed

intracellular and intercellular ROS signaling play protective roles in the UPR-associated

cell death.

74

A. B

.

β Protein is in the ER and Co-Fractionates with BiP.

0

10

20

30

40

50

0 1 2 3 4 5 6 7

Time (days)

% Io

n Le

akag

e

wt atrbohD

G

Gα1 has been immunolocalized to the plasma membrane and endoplasmic reticulum

before (Weiss et al., 1997). As shown in Chapter 4, the interaction between Gα1 and

Gβ1γ1 complex in Arabidopsis mesophyll protoplasts were identified using fluorescence

resonance energy transfer (FRET) and co-immunoprecipitation. Given the evidence that

the Gβ protein is involved in the ER stress response, we asked whether the cellular

localization is consistent with such a role. A CFP-Gβ fusion construct was transformed

into agb1-2 mutant plants and found to complement its mutant phenotype, as judged by

leaf morphology. Expression of the CFP-Gβ was controlled by its native promoter to

Col-O atrobhD

atrobhF atrobhD/F

Col-O atrobhD

atrobhF atrobhD/F

atrbohFatrbohD/F

Figure 10. Tm-induced cell death was accelerated in atrobhD and atrobhD/F double mutant plants. A. Leaves of 5 week old wildtype (Col-O) plants, atrobhD, atrobhF and atrobhD/F mutant plants were injected with 15 μg/ml Tm and photographs were taken after 5 days. B. Cell damage was monitored by the release of electrolytes from leaves 3 days to 7 days following the injection.

75

ensure the same expression level of Gβ in transgenic plants as wildtype plants. A leaf

extract was prepared and subjected to ultracentrifugation to obtain the microsomal

fraction, which was then further purified by aqueous two-phase partitioning into plasma

membrane and intracellular membrane fractions. The intracellular membrane fraction was

further subjected to sucrose density gradient centrifugation to fractionate the Gβ protein.

Both Gα and Gβ proteins were enriched in the microsomal fraction (Fig. 11A). The Gα

protein was present at a slightly higher level in the plasma membrane than in the

intracellular membrane fraction. In contrast, a majority of the Gβ protein was in

intracellular membranes, although a significant fraction of it co-fractionated with the Gα

protein in the plasma membrane (Fig. 11A). Thus, the Gβ protein was mainly associated

with the ER, as judged by its co-sedimentaion with the luminal marker protein BiP in the

sucrose gradient, while the Gα protein equally distributed throughout the sucrose gradient

(Fig. 11B). The accumulation of the Arabdiopsis Gβ protein in ER suggested that it may

have a signaling function in the ER.

Gβ Protein is Degraded during the UPR.

ER-associated protein degradation is another hallmark of the UPR. To evaluate the

degradation of Gβ protein during the UPR, we monitored the amount of Gβ protein over

the course of Tm treatment. The abundance of Gβ protein started to decline on the first

day after Tm treatment and continuously decreased to roughly 25% of its initial level by 4

days after Tm treatment (Fig. 12). In contrast, the amount of Gα protein did not decrease,

76

but rather showed a slight increase over the course of the 3 days following Tm treatment

(Fig. 12). Hence, the decreased level of Gβ protein in the course of Tm treatment

suggested that Gβ protein underwent ER-associated degradation during the Tm-induced

UPR. The differential changes in Gα and Gβ protein levels in response to Tm further

suggeste that the Arabidopsis Gα and Gβ proteins have functions separable from those of

the heterotrimeric G protein.

T S M U L

anti-GPA

anti-CFP

anti-BiP

T S M U L

anti-GPA

anti-CFP

anti-BiP

T S M L UA.

B.

Figure 11. Gβ protein is localized in the ER and co-fractions with BiP. A. Aliquots of total cell extracts (T), soluble (S) supernatant and pelleted microsomal (M) fractions, as well as lower, intracellular membrane (Im) fractions and the upper, plasma membrane fraction (Pm) after aqueous two-phase partitioning were analyzed together by SDS-PAGE and Western blotting using anti-GPA antibodies to detect Gα protein, anti-CFP antibodies to detect the CFP-Gβ fusion protein in extracts of transgenic plants expressing a Gβ-CFP fusion protein from the Gβ promoter and anti-BiP antibodies for the ER markers BiP1. B. The Im fraction was sedimented through a 10-50% sucrose density gradient and aliquots of each fraction were analyzed by SDS-PAGE and Western blotting as in A.

anti-GPA

anti-CFP

anti-Bip

% Sucrose

anti-GPA

anti-CFP

% Sucrose

anti-Bip

anti-GPA

anti-CFP

anti-Bip

77

Tm 0 1 2 3 4 agb1-2

anti-GPA

anti-CFP

Gβ and Gα proteins were detected as in Figure 11. in total extracts from Gβ-CFP transgenic plants injected with Tm at 30 μg/ml and sampled after 1-4 days.

Figure 12. Gβ protein is degraded during the UPR.

Discussion

We have presented evidence that both signaling by the Gβ subunit of the

Arabidopsis heterotrimeric G protein and ROS signaling are implicated in the

UPR-associated cell death in response to Tm treatment. Gβ null mutant plants are much

more resistant to Tm-induced cell death than either wildtype or Gα null mutant plants.

This observation indicates that the Gβ subunit, but not the Gα subunit, plays a direct role

in UPR-associated cell death. The cell death induced by Tm is accelerated by the

application of DPI and NAC. Moreover, a mutation in the AtrbohD gene also exacerbates

Tm induced cell death, which indicates that intracellular and intercellular ROS signaling

play a protective role in UPR-associated cell death. The transcriptional response of Gβ

mutant plants to Tm is much less pronounced than that of wildtype plants, which

indicates that the Gβ subunit is directly involved in the UPR-associated gene expression.

The administration of DPI delays the activation of the UPR-associated gene expression,

78

which indicates that ROS is required for the rapid activation of UPR-associated gene

expression.

A significant fraction of the Arabidopsis Gβ protein is associated with ER membranes,

indicating that the Gβ subunit may function directly in ER stress. Gβ protein is degraded

during the UPR, presumably by ER-associated protein degradation, while Gα protein is

not. Given the resistant phenotype of agb1-2 mutants to Tm induced cell death and the

presence of a majority of the Gβ subunit in the ER, we conclude that signaling events

mediated by Gβγ subunit, not Gα subunit, trigger UPR-associated cell death, which

further supports the concept that Gβγ and Gα subunits serve independent functions in

different biological processes.

Tm has been used widely to study the UPR in mammalian cells (Shen et al., 2004).

Using Tm, we have demonstrated that ER stress can cause leaf senescence in plants.

Senescence, as the last developmental stage of plants, is defined as a specialized plant

programmed cell death (PCD) (Gepstein, 2004). It is well documented that ER stress can

activate PCD in mammals to eliminate severely damaged cells (Shen et al., 2004; Wu and

Kaufman, 2006), which suggests that PCD induced by ER stress is conserved in plant and

animal systems. The phenotypic characterization of cell death in Arabidopsis plant leaves

during Tm-induced ER stress could be used as a tool to further evaluate and identify the

essential components in plant UPR.

Thapsigargin (Tg), which depletes the cell’s energy stores for protein folding

(Thastrup et al., 1990), and dithiothreitol (DTT) (Jamsa et al., 1994), which creates

79

reductive stress in the ER, were also tried to induce plant ER stress. We found that their

effects are different from that of Tm with respect to leaf senescence. DTT caused leaf

necrosis quickly after the injection (data not shown) and Tg induced only slight leaf

senescence (data not shown) at hundreds of times higher concentrations than used in

mammalian cells. This may be due to the impermeability of plant cells to this compound.

Previous studies have shown that the Arabidopsis Gβ protein plays important roles in

root growth (Ullah et al., 2003), leaf development (Lease et al., 2001), ozone stress

response (Joo et al., 2005) and pathogen response (Llorente et al., 2005). The results of

the present study establish a connection between signaling by the Gβγ complex and the

UPR. Although several downstream effectors of the Gα subunit have been identified,

such as phospholipase Dα in Arabidopsis (Zhao and Wang, 2004) and the small GTPase

Rac1 in Rice (Suharsono et al., 2002), no downstream targets of Gβγ signaling have yet

been identified in plants. Moreover, there are no other proteins or signaling identified to

be involved in plant UPR. Thus, it is hard to predict how Gβγ signaling mediates the UPR

in Arabidopsis.

Gβγ signaling is better understood in animals than in plants. Some signaling pathways

mediated by Gβγ are conserved in animals and plants (Assmann, 2002). Hence

knowledge for animals may give some hints for the role of Arabidopsis Gβγ signaling in

the UPR. In animal cells, Gβγ is a central component of phosphoinositide signaling

through its ability to activate phospholipase Cβ2 (PLCβ2) and PLCβ3 to generate the

second messengers diacylglycerol and inositol (1,4,5) triphosphate (IP3), which further

80

interact with receptors to trigger Ca+2 release from the ER (Feng et al., 2005; Bonacci et

al., 2006). This signaling pathway which originates from Gβγ to IP3, resulting in Ca+2

release is involved in many physiological responses, such as ER stress responses,

apoptotic and necrotic cell death (Lin et al., 1997; Kim et al., 2001; Xu et al., 2001).

Nonetheless, whether this signaling pathway is involved in UPR-associated cell death in

animal cells has not been directly investigated. Although the connection between the Gβγ

complex, IP3 production and Ca+2 release has been established in Arabidopsis, PLC

activation, IP3 production, and Ca+2 spikes are all rapid responses to some biotic and

abiotic stresses in Arabidopsis (Price et al., 1994; Knight et al., 1996, 1997). Moreover,

Ca+2 release is unequivocally involved in programmed cell death triggered by different

stresses in Arabidopsis (Davies et al., 2006; Ali et al., 2007). Hence, it is possible that

PLC or perhaps ER IP3 receptors are Gβγ complex effectors that contribute to Ca+2

releases to mediate UPR-associated cell death in Arabidopsis.

As important a signaling molecule as calcium, ROS also plays critical roles in

Tm-mediated plant ER stress response. Oxidative stress and ER stress have been

investigated separately, although there is increasing evidence that they are interconnected

with respect to cellular environment and stress responses (Fedoroff, 2006). ROS may

function in one of the following ways to regulate ER stress response. First, the ER strictly

requires a reducing environment for protein folding, disulfide bond formation and some

post-translational protein modifications. Any perturbation of this reducing environment in

the ER prevents proper folding of new synthesized proteins. Thus, excess ROS could

81

cause ER stress or aggravate ER stress (Schroder and Kaufman, 2005). However, our

results show that elimination of ROS exacerbates Tm-induced cell death, indicating that

ROS serve a protective role in the normal UPR in Arabidopsis.

On the other hand, ER stress can disrupt the electron transfer chain in protein

disulfide bond formation, which results in the generation of ROS (Schroder and Kaufman,

2005). ROS could act on chaperone proteins to regulate their biological activity (Jakob et

al., 1999; Papp et al., 2003). Heat shock factor (Hsf1) belongs to a highly conserved

eukaryotic transcription factor family, which induces the expression of a variety of heat

shock proteins in response to heat, oxidative stress and a variety of other stresses. The

DNA binding activity of Hsf1 is directly promoted by oxidation with H2O2

(Jacquier-Sarlin and Polla, 1996; Mishra et al., 2002). Arabidopsis contains heat shock

factors, some of which have been identified as potentially redox-regulated (Baniwal et al.,

2004). Activation of chaperone proteins by ROS could explain the effect of ROS

scavengers on the acceleration of cell death induced by Tm because suppressing ROS

concurrently deactivates chaperone proteins, resulting in less protective responses to ER

stress. However, some ER stress related proteins, such as ATF6, are activated by

reduction instead of oxidation (Nadanaka et al., 2007). It is hard to conclude that total

activity of chaperones is higher in oxidative condition or reducing condition. Thus, ROS

may also act on other signaling pathways to manipulate cell death-associated UPR in

Arabidopsis.

There is an apparent paradox between the protective role of ROS in cell death

82

associated UPR based on our observation and long-held belief that ROS initiated or

spreaded cell death in the pathogen and ozone response. In previous studies, either in the

oxidative stress response or pathogen response in Arabidopsis, extracellular ROS

produced by the AtrbohD- and AtrbohF-encoded NADPH oxidases contributed to cell

death, since mutation of AtrbohD and AtrbohF rendered resistance to both responses

(Torres et al., 2002; Joo et al., 2005). However, a recent study showed that cell death in

null mutant plants with lsd1 mutation, which was identified by its “runaway cell death,”

phenotype in oxidative stress to superoxide (Jabs et al., 1996), was exacerbated by the

mutation of AtrbohD and curtailed by overexpression of AtrbohD in pathogen response

(Torres et al., 2005), suggesting ROS can play a protective role, limiting the spread of cell

death in the pathogen response. However, how ROS signals are integrated and able to

commit cells to survival or death individually is not clear yet.

83

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Chapter 4

CHARACTERIZATION OF THE ARABIDOPSIS

HETEROTRIMERIC G PROTEIN COMPLEX

Abstract

Using fluorescence resonance energy transfer (FRET) and co-immunoprecipitation,

we show interaction between the β1 and γ subunits and between the α1 and β1γ subunits

of the Arabidopsis heterotrimeric G proteins in plant mesophyll protoplasts. We report

that both the Gα and the Gβ subunits are associated with macromolecular complexes in

plasma membrane fractions. We estimate the size of the G protein containing plasma

membrane complex to be approximately 669 kD based on blue native polyacrylamide gel

electrophoresis, suggesting that the heterotrimeric G protein is present in a complex with

other proteins. In plants homozygous the agb1-2 Gβ1 null allele, Gα1 is present in both

the large ca. 669 kD complex and in smaller complexes. Deletion of the Gβ1 interacting

domain in Gα1, ahGα1, abolishes normal function of Gα1 in the oxidative stress response

to ozone, suggesting the interaction between Gα1 and Gβ1γ complex may be required for

transmitting the ozone signal in plant cells. Activation of Gα1 subunit by GTPγS does not

result in complete dissociation Gα1 subunit from its complex. Treatment of the plasma

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membrane fraction with hydrogen peroxide (H2O2), an important signaling molecule,

results in the partial dissociation of the Gα1 complex, suggesting direct or indirect

activation of G-protein signaling by ROS in the oxidative stress response.

Introduction

The results of both pharmacological and genetic studies indicate that the plant

heterotrimeric G protein is involved in the transmission of different kinds of signals,

including light (Warpeha et al., 1991) and hormones (Bowler et al., 1994), as well as in

the regulation of ion channels (Wu and Assmann, 1994). The Arabidopsis gpa1 mutant,

which lacks the Gα1 protein (AtGPA1), exhibits reduced cell division during hypocotyl

and leaf formation (Ullah et al., 2001), while overexpression of AtGPA1 causes ectopic

cell division, including massive meristem proliferation (Okamoto et al., 2001).

Homozygous gpa1 mutant plants show reduced sensitivity than wildtype plants to ABA

inhibition of stomatal opening and inward K+ channel regulation (Wang et al. 2001),

although gpa1 mutant seeds exhibit wild-type sensitivity to ABA inhibition in

germination (Ullah et al., 2002; Pandey and Assmann, 2004). In addition, gpa1 mutant

plants are not only less sensitive to gibberellic acid, but completely insensitive to

brassinosteroids (BR) (Ullah et al., 2002). Arabidopsis agb1-2 mutant plants, which lack

the Gβ protein (AtAGB1), show alterations in leaf, flower and fruit development as well

as cell division in the hypocotyl and are D-glucose hypersensitive (Lease et al., 2001;

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Chen et al., 2006; Wang et al., 2006).

Evidence is also accumulating that the heterotrimeric G protein plays a role in the

plant response to bacterial and fungal pathogens, as well as abiotic stress. Heterotrimeric

G protein signaling to membrane-bound NADPH oxidase has been implicated in the

development of disease resistance and in the apoptotic hypersensitive response in rice

(Suharsono et al., 2002). A signal perception role of heterotrimeric G protein in

necrotrophic fungus responses is also suggested by the enhanced susceptibility phenotype

of agb1-1 mutant plant to Plectosphaerella cucumerina (Llorente et al., 2005). Moreover,

it was reported that the Gα1 and Gβ1 subunits serve both separable and synergistic

functions in signaling by reactive oxygen species (ROS) in the oxidative stress response

(Joo et al., 2005). An independent role for the Gβ1γ complex of the heterotrimeric G

protein in cell death signaling in the unfolded protein response has been reported in

Arabidopsis.

Contrary to the large number of different G protein subunits in animals, Arabidopsis

has only one Gα protein (Ma et al., 1990), one Gβ protein (Weiss et al., 1994), and at

most two Gγ proteins (AtAGG1 and AtAGG2) (Mason and Botella, 2000, 2001). All of

them exhibit limited homology with their animal counterparts. The AtGPA1 protein is

roughly 30% identical with the mammalian Gα subfamily protein, AtAGB1 shows about

42% similarity to mammalian Gβ subunits, and AtAGGs display 24%-31% identity with

certain mammalian Gγ subunits (Mason and Botella, 2001). At the cellular level, Gα1 has

been immunolocalized to the plasma membrane and endoplasmic reticulum (Weiss et al.,

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1997), while Gβ1 has been detected in the plasma membrane, endoplasmic reticulum and

Golgi apparutus (Obrdlik et al., 2000, Adjobo-Hermans et al., 2006). Interactions between

Gβ1 and both Gγ1 and Gγ2 have been detected by yeast two-hybrid and in vitro binding

assays (Mason and Botella, 2000, 2001), and there is evidence from yeast two-hybrid and

co-immunoprecipitaion assays for interactions between the rice Gα and Gβ subunits

(Kato et al., 2004). Structural predictions for Gα1, Gβ1 and Gγ1 suggest that they can form

a heterotrimer similar to that formed by mammalian G protein subunits (Ullah et al.,

2003). Although the heterotrimerization of Arabidopsis G protein subunits in cowpea

protoplasts was recently reported (Adjobo-Hermans et al., 2006), there is still limited

information available about the structural and biochemical characteristics of Arabidopsis

heterotrimeric G protein.

Here we provide structural and biochemical evidence that further supports the

concept of common and separate signaling functions of Gα1 and the Gβ1γ1 complex. First,

we show interactions between Gβ1 and Gγ1, as well as between Gα1 and Gβ1γ1 complex

in Arabidopsis mesophyll protoplasts using fluorescence resonance energy transfer

(FRET) and co-immunoprecipitation, presenting direct evidence that the plant G protein

subunits form a heterotrimer in vivo. Then, we show that the heterotrimeric G protein is

part of a large complex using blue native gel electrophoresis. We also show that Gα1 is

presenting a complex that is more than 669 kD and found in the plasma membrane

fraction in wild type and agb1-2 mutant plants, although some smaller complexes are also

detected in the agb1-2 mutant. We further show that a mutant ahGα1 lacking Gβ1

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interacting domain in Gα1 is not functional in the oxidative stress response to ozone.

Although activation by GTPγS can not completely dissociate Gα from the complex, the

Gα complex dissociates upon treatment with hydrogen peroxide (H2O2), suggesting a

mechanism by which G protein signaling can be directly or indirectly activated in

response to oxidative stress.

Methods and Materials

Plant Materials

We used Arabidopsis thaliana Col-0 plants and agb1-2 and gpa1-4 null mutant

homozygotes in the Col-0 background (Ullah et al., 2003). Plants used to isolate plasma

membrane fractions were grown in MetroMix 200 (Scotts-Sierra Horticultural Products

Company, Marysville, OH) in 5-cm pots (51 per flat) at 65% humidity under fluorescent

light at 30 W/m2/s with a 14-h-light/10-h-dark photoperiod for 5 weeks.

Generation of Vectors for Expression of Green and Yellow Fluorescent Protein-G Protein

Fusions

AtAGB1 and AtAGG1 were cloned by PCR from an Arabidopsis thaliana Col-0

cDNA library, which was made as previously described (Han et al., 2004). According to

the sequence information in the Arabidopsis database (http://www.arabidopsis.org),

primers were designed as follows, with the underlined sequences adding restriction sites

and the bold faced sequences adding a spacer (purchased from Integrated DNA

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Technologies, Coralville, IA). The forward primer for AtAGB1 is:

(5′-AGATCTGGAGGTGGAGGTAGTATGTCTGTCTCCGAGCTCAA-3′). The

reverse primer for AtAGB1 is: (5′-TCTAGATCAAATCACTCTCCTGTGTCC-3′). The

forward primer for AtAGG1 is: (5′-AGATCTGGAGGTGGAGG TAGTATGCGAGA

GGAAACTGTGGTT-3′). The reverse primer for AtAGG1 is:

(5′-TCTAGATCAAAGTATTAAGCATCTGCA-3′). To insert CFP into AtGPA1, the

binary vector of loop CFP-AtGPA1 was requested from Dr. Alan Jones (University of

North Carolina, Chapel Hill, North Carolina). The full length sequence of AtGPA1(l)CFP

was cloned from the binary vector. The amplified PCR fragments were cloned into

pGEM-Teasy (Promega, Madison. WI) and sequences were verified. To make constructs

of pVAV321-35S-(C/Y)FP-AtAGB1 and pVAV321-35S-YFP- -AtAGG1, PCR products

were excised from pGEM-Teasy (Promega) and further cloned into pVAV321-35S-C/YFP

(a kind gift of Dr. Xuemei Chen, University of California, Riverside, California) using the

BglII and XbaI sites. To make constructs pVAV321-35S-AtGPA1(l)CFP,

pVAV321-35S-AtAGB1 and pVAV321-35S-AtAGG1, PCR fragments of AtGPA1(l)CFP,

AtAGB1 or AtAGG1 were excised from corresponding pGEM-Teasy constructs and

cloned into pVAV321 with the removal of C/YFP via NcoI and XbaI. Then, the entire

expression cassette of 35S-AtAGG1 or 35S-AtAGB1 was ligated into

pVAV321-35S-YFP-AtAGB1 or pVAV321-35S-YFP-AtAGG1 individually using the

SmaI and ClaI sites, which were modified by Klenow large fragment, resulting in

pVAV321-35S-AtAGG1-35S-YFP-AtAGB1 and pVAV321-35S-AtAGB1-35S-

94

YFP-AtAGG1. Finally, the entire expression cassette of 35S-AtGPA1(l)CFP was cloned

into pVAV321-35S-AtAGG1- 35S-YFP-AtAGB1 or pVAV321-35S-AtAGB1-35S-YFP-

AtAGG1 via SmaI and SacI sites, which were modified by Klenow large fragment,

resulting in pVAV321-35S-AtGPA1(l)CFP-35S-AtAGG1-35S-YFP-AtAGB1 and

pVAV321-35S-AtGPA1(l)CFP-35S-AtAGB1--35S-YFP-AtAGG1. The 35S-AtGPA1(l)

CFP and 35S-YFP-AtAGB1 expression cassettes were further cloned into the

pCAMBIA3300 Agrobacterium binary vector and transformed into homozygous agb1-2

and gpa1-4 mutant plant individually. Transformed plants that exhibited the wildtype

phenotype in terms of leaf morphology on 10 g/ml glufosinate MS plates were used for

further study (Ullah et al., 2003).

Protoplast Isolation and Transfection, Confocal Microscopy and FRET

Arabidopsis mesophyll protoplasts were prepared from fully expanded leaves of 4

to 5 week old plants and transfected by a polyethyleneglycol method as described by J.

Sheen’s laboratory http://genetics.mgh.harvard.edu/sheenweb/prototcols. An additional

21% sucrose gradient was applied to the isolated protoplasts to improve the quality of

transfected protoplasts, followed by the transfection with plasmids containing the FRET

pair to be tested. Plasmids were isolated using a Plasmid Maxi Kit (Qiagen, Chatsworth,

CA). A total of ~1x105 cells were transfected with 50 µg plasmid for each microscopy

observation, FRET measurement or immunoprecipitation experiment. The transfected

protoplasts were incubated at 22°C for 12-16 h before being mounted in chambers

95

(Molecular Probes, Eugene, OR) for microcrospy. The transfected protoplasts were

imaged using a Zeiss laser scanning microscope (Carl Zeiss, Thornwood, NY), LSM 510

META with a 40×NA 1.2 water objective. To monitor fusion protein expression and

localization, protoplasts were excited with two Argon laser lines, 458 nm for CFP and

514 nm for YFP and emission images were collected simultaneously with 480-520 nm

filter for CFP and 530-590 nm filter for YFP.

FRET is a widely used method to identify interaction of two proteins tagged with a

FRET donor (CFP) and acceptor (YFP) fluorophore pair in vivo. FRET between CFP and

YFP fusion G protein subunits was measured with a Zeiss LSM510 Meta spectral

confocal fluorescence microscope (Carl Zeiss). The transfected protoplasts were excited

with a chameleon multiphoton laser (Coherent MRU 1000), which was modulated to 820

nm wavelength and set at 4-5% laser intensity ideal for cross-talk free FRET analysis.

The emission spectra from selected regions (ROI) were recorded by a connected

multi-channel-spectrometer (MCS) in twelve channels, each with a 10 nm band width,

from 464 to 584 nm, using a 650 KP dichroic mirror in the lambda stack acquisition

mode. Emission spectra were recorded from 20 individual protoplasts transfected with

each G protein FRET pair tested. Spectral analysis with automatic peak detection and

spectral color encoding, were done with the software package for the LSM 510. Final

florescence spectra of G protein subunits CFP and YFP fusion protein were corrected for

background florescence. The reliability test in spectrum-based FRET measurement with

2-photon excitation was performed using published negative and positive FRET pair

96

fusion protein in mesophyll protoplasts (Kato et al., 2002). FRET tested pairs of CFP and

YFP fusion protein constructs were kindly provided by Dr. Lam Eric.

Immunoprecipitation and Western blotting

The catch and release reversible immunoprecipitation system (Upstate, Lake Placid,

NY) was used for immunoprecipatation experiments. The transfected protoplasts were

lysed with 1×wash buffer provided by the kit and then centrifuged at 15,000×g for 10 min.

The clear extract was incubated with the rabbit polyclonal anti-Gα antibody (1:250) and

the affinity ligand (1:50) in the kit column at 4°C for 10-12 h. The column was washed

three times by centrifugation with 1×wash buffer. 1×Denaturing elution buffer in the kit

was used to elute proteins binding in the column, which were subjected to further

electrophoresis and Western blotting. Total extract, flow through that was concentrated

with Strataclean resin (Stratagene, La Jolla, CA), and eluted proteins were loaded on a

12% polyacrylamide discontinuous gel (Bio-Rad mini electrophoresis system, Hercules,

CA). After electrophoresis, proteins were transferred to Hybond-P PVDF membrane

(Amersham, Piscataway, NJ) using a Mini Trans-Blot electrophoretic transfer cell

(Bio-Rad). Immunoblotting was performed with mouse monoclonal anti-CFP antibodies

(1:2000, BD Bioscience, Mountain View, CA). After incubation with horseradish

peroxidase–conjugated anti-rabbit IgG antibodies, proteins were detected using ECL Plus

protein gel blotting detection reagents (Amersham) according to the manufacturer’s

instructions.

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Blue Native (BN)-PAGE

Plasma membrane fraction for BN-PAGE was prepared in an aqueous two-phase

partitioning system as described (Thomson et al., 1993; Yan et al., 1998; Sagi and Fluhr,

2001). Blue Native/SDS-PAGE 2-dimensional gel electrophoresis was performed as

described previously with some modification (Rivas et al., 2002; Han et al., 2004;

Karlova et al., 2006). Plasma membrane proteins were incubated with solubilization

buffer (20 mM Bis-Tris-HCl, pH 7.0, 250 mM ε-aminocaproic acid, 2 mM EDTA, 1.0%

Triton X-100, 0.25% Coomassie blue G 250 and 10% glycerol) for 30 mins and

centrifuged twice at 15,000×g for 5 mins. Solubilization buffer containing 100 µM

GTPγS or 20µM H2O2 was used for GTPγS or H2O2 treatment experiments. The

supernatant was separated in 5.5%-16% polyacrylamide gradient gel and albumin bovine

monomer (66 kD), lactate dehydrogenase (140 kD), catalase (232 kD), ferritin (440 kD),

porcine thyroid (669kD) from Amersham, were loaded alongside as marker proteins. 1st

dimension BN-PAGE gel was transferred to Hybond-P PVDF membrane (Amersham)

and immunoblotted with anti-G/C/Y antibodies (BD Bioscience) to detect CFP tagged

Gβ1 complex (anti-G/C/Y antibodies detect GFP, CFP and YFP proteins). The remaining

first dimension lanes of the BN-PAGE gel were cut and incubated in denaturing solution

(1% SDS, 1% β-mercaptoethanol) for 2h and mounted on 10% SDS-PAGE gel. After

electrophoresis, the gel was subjected to immunoblotting analysis with anti-Gα antibody,

which can not detect the protein complex in 1st dimension BN-PAGE gel.

98

Mutagenesis Study in AtGα1

We amplified ahAtGPA1 and AtGPA1 by PCR from an Arabidopsis cDNA library as

described above. For ahAtGPA1, the forward primer is (5′-CACCATGGGCTTACTC

TGCAAGCAAAGGCTGAAAAGCATATT-3′) and the reverse primer is

(5′-TCATAAAAGGCCAGCCTCCAGTAA-3′). For AtGPA1, the forward primer is

(5′-CACCATGGGCTTACTCTGCAGTAGAA-3′) and the reverse primer is

(5′-TCATAAAAGGCCAGCCTCCAGTAA-3′). PCR products were subcloned into the

pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA), then cloned into Gateway plant

transformation destination binary vector pGWB2 (Karimi et al., 2002) by LR

recombination reactions. Both 35S:GPA1 and 35S:ahGPA1 constructs were transformed

into gpa1-4 mutant backgrounds. The transformants were compared with wildtype plants

for their sensitivity to ozone.

Recombinant AtGPA1 Expression, Purification and [35S]GTPγS Binding Assay

We amplified AtGPA1 by PCR from an Arabidopsis cDNA library as described

above with forward primer (5′-GAGCTCATGGGCTTACTCTGCAGTAGAA-3′) and

reverse primer (5′-GGTACCTCATAAAAGGCCAGCCTCCAGTAA-3′), cloned it into

QIAexpress pQE-30 RGS•His tag vector (Qiagen) at the SacІ and KpnІ restriction sites

(underlined in primer sequences) and expressed it in BL21-CodonPlus (DE3)-RIPL strain

(Statagene). The recombinant 6×His tagged Gα1 was purified according to the

99

manufacturer’s instructions (The QIAexpressionist, Qiagen). Purification was confirmed

by PAGE and the purified recombinant AtGPA1 protein was dialysed against GTPγS

binding assay buffer (50 mM Tris•HCl, pH 8.0, 5 μM GDP and 3 mM MgCl, 1 mM

EDTA, 100 mM NaCl). The recombinant Gα1 was incubated with 20 nM [35S]GTPγS in

the above buffer for different times and the reaction was diluted with stop solution (25

mM Tris•HCl, pH 8.0, 25 mM MgCl, 100 mM NaCl). The reaction was further applied to

a 0.45 μm nitrocellulose membrane filter (Whatman, Florham Park, NJ), which was

rinsed 3 times with stop solution. The amount of recombinant Gα1 bound [35S]GTPγS

was then quantified by liquid scintillation spectrometry. The specific binding of

[35S]GTPγS with recombinant Gα1 was evaluated by nucleotide competition experiments.

The effect of H2O2 on the binding of [35S]GTPγS to recombinant Gα1 was tested as

described (Nishida et al., 2002).

Results

FRET Detection of In Vivo Interactions between the β1 and γ1 Subunits of the

Arabidopsis Heterotrimeric G protein.

First, we used FRET to determine the interaction between Gβ1 and Gγ1 in vivo.

Modeling predictions suggested the existence of the heterotrimeric form of G protein

subunits in Arabidopsis (Fig. 1A) (Ullah et al., 2003). Moreover, interaction between Gβ1

and Gγ1 has been demonstrated in yeast two-hybrid and in vitro pull-down assays (Mason

100

and Botella, 2000). In yeast and leukocytes, FRET has been used to monitor the

association of the subunits of the heterotrimeric G protein and the dissociation of the Gα

subunit from Gβ1γ1 dimer in response to external stimuli (Janetopoulos et al., 2001;

Ruiz-Velasco and Ikeda, 2001; Humrich et al., 2005). Therefore, we used FRET to

determine whether the Arabidopsis G protein subunits form the heterotrimer in vivo.

A. B.

35S TL NOSAtGPA1(l)CFP

35S TL NOSYFP-AtAGB1

35S TL NOSYFP-AtAGG1

35S TL NOSAtGPA1(l)CFP

35S TL NOSYFP-AtAGB1

35S TL NOSYFP-AtAGG1

35S TL NOSAtAGG1 35S TL NOSYFP-AtAGB1

35S TL NOSAtAGB1 35S TL NOSYFP-AtAGG1

35S-AtGPA1(l)CFP-NOS 35S-YFPAtAGB-NOS35S-AtAGG-NOS

35S-AtGPA1(l)CFP-NOS 35S-YFPAtAGB-NOS35S-AtAGG-NOS

35S TL NOSAtAGG1 35S TL NOSYFP-AtAGB1

35S TL NOSAtAGB1 35S TL NOSYFP-AtAGG1

35S-AtGPA1(l)CFP-NOS 35S-YFPAtAGB-NOS35S-AtAGG-NOS

35S-AtGPA1(l)CFP-NOS 35S-YFPAtAGB-NOS35S-AtAGG-NOS

C. D.

0

40

80

120

160

200

450 490 530

Wavelength (nm)

L-CFP-Ga

Background

YFP-Gß

YFP-Gγ1

Fluo

resc

ence

Inte

nsity

(a

rbitr

ary

units

)

5700

40

80

120

160

200

450 490 530

Wavelength (nm)

L-CFP-Ga

Background

YFP-Gß

YFP-Gγ1

Fluo

resc

ence

Inte

nsity

(a

rbitr

ary

units

)

570

L-CFP-Gα

YFP-Gβ

YFP-Gγ1

101

A, The predicted structure of Arabidopsis heterotrimeric G protein is depicted (Ullah et al., 2003) B, Constructs of CFP and YFP of Arabidopsis G protein subunits were used in this study for FRET measurement. C, Cellular localization images of CFP and YFP of Arabidopsis G protein subunits were taken. CFP was excited by chameleon multiphoton laser modulated to 820 nm wavelength and YFP was excited by Ar laser lines at 514 nm. D, Fluorescent emission spectra from selected regions of C (crossing marks) were recorded by Zeiss LSM510 Meta spectral confocal fluorescence microscope.

Figure 13. Spectral characteristics and cellular localization of CFP and YFP of Arabidopsis G protein subunits

Based on the Arabidopsis G protein structural predictions, we constructed genes

encoding N-terminal fusions of enhanced C/YFP (yellow/cyan fluorescent proteins) with

Gβ1 and Gγ1 and expressed them from a CaMV 35S promoter (Fig. 13A and 13B). We

transfected Arabidopsis mesophyll protoplasts with YFP-Gβ1 and CFP-Gγ1 and

determined the cellular localization by microcopy and their interaction by FRET. We

observed the fluorescence of CFP-Gβ1 in small bodies (Fig. 13C) in the cytoplasm. The

fluorescence of YFP-Gγ1 was observed at the very periphery of the protoplast, which

indicates that Gγ1 is predominantly localized in the plasma membrane (Fig. 13C). Upon

co-transfection of CFP-Gβ1 and YFP-Gγ1, we observed signal from CFP-Gβ1 at the very

periphery of the protoplast along with YFP-Gγ1 (Fig. 14A.1), suggesting that plasma

membrane localization of Gβ1 requires the co-expression of Gγ1. We also used the

co-transfected protoplasts with CFP-Gβ1 and YFP-Gγ1 to determine FRET by spectral

recording microscopy with crosstalk-free chameleon multiphoton laser excitation. FRET

102

is detected as the occurrence of an emission spectral shift from CFP to YFP (Fig. 14B). In

protoplasts co-transfected with CFP-Gβ1 and YFP-Gγ1, we detected a strong increase

from 520 to 530 nm, representing the typical YFP spectrum (Fig. 13D), opposed to CFP

spectrum itself (Fig. 13D), which has a broad peak from 475 to 500 nm, indicating that

Gβ1 and Gγ1 interact in plant cells.

FRET Detection of the Interaction between the α1 Subunit and the β1γ1 Complex.

Next, we used FRET to determine the interaction between Gα1 and Gβ1γ1 in vivo.

To minimize the possibility of interference of CFP with the structure and function of the

Gα1 (Humrich et al., 2005), we inserted CFP into the putative αAB-loop within the α

helical domain of Gα1 as described previously (Fig. 13A) (Chen et al., 2003). We

co-transfected Arabidopsis mesophyll protoplasts with combinations of plasmids

encoding the following: CFP(l)-Gα1 and YFP-Gβ1; CFP(l)-Gα1 and YFP-Gγ1; and

CFP(l)-Gα1, YFP-Gβ1 and YFP-Gγ1. We were able to detect the spectral shift only when

we co-transfected the protoplasts with constructs containing the coding sequences for all

three subunits, not just two of the three (Fig. 14A. 2, 3 & 4). This observation indicates

that although FRET pairs are transfected into wildtype protoplasts which contain

endogenous Gγ1 or Gβ1, for the FRET detection, overexpression of Gγ1 or Gβ1 is still

needed to ensure plasma membrane distribution of the Gβ1 subunit by Gγ1 or closeness of

Gγ1 to Gα1 by Gβ1. This observation further suggests that the α subunit does not interact

with either the β or the γ subunit alone in Arabidopsis protoplasts (Fig. 14A. 4 and 14B).

103

To further determine whether the energy transfer is from CFP(l)-Gα1 to YFP-Gβ1 or

from CFP(l)-Gα1 to YFP-Gγ1 in the G protein complex formed in protoplasts, we created

a single construct expressing all three genes with two as a FRET pair and the third one

untagged as follows: 35S-CFP(l)-Gα1-35S-YFP-Gβ1-35S-Gγ1 and 35S-CFP(l)-Gα1-

35S-Gβ1-35S-YFP-Gγ1 (Fig. 13B). We detected the emission spectral shift in protoplasts

transfected with 35S-CFP(l)-Gα-35S-Gβ1-35S-YFP-Gγ1 (Fig. 14A. 5, 14A. 6 and 14B),

but not in protoplasts transfected with the 35S-CFP(l)-Gα1- 35S-YFP-Gβ1-35S-Gγ1

construct. Thus, energy transfer occured between CFP(l)-Gα1 and YFP-Gγ1, but not

between CFP(l)-Gα1 and YFP-Gβ1, indicating that Gα1 subunit is in closer proximity to

the Gγ1 subunit than to Gβ1 subunit in the G protein complex formed in Arabidopsis

protoplasts. However, energy transfer between Gα1 and Gγ1 does not occur in the absence

of the Gβ1, suggesting that the subunits of Arabidopsis heterotrimeric G protein form a

complex in vivo.

A.

CFP-Gβ1+YFP-Gγ1 YFP-Gβ1+L-CFPGα1 YFP-Gγ1+L-CFPGα1

L-CFPGα1+YFP-Gβ1+YFP-Gγ1 L-CFPGα1-Gβ1-YFP-Gγ1 L-CFPGα1-N-YFPGβ1-Gγ1

1 2 3

4 5 6

104

B.

0

0.4

0.8

1.2

1.6

2

450 490 530 570wavelength (nm)

YFP

/CFP

CFP-Gß1+YFPGγ1

YFP-Gß1+ L-CFPGa1YFP-Gγ1+ L-CFPGa1

0

0.4

0.8

1.2

1.6

2

450 490 530 570wavelength (nm)

YFP

/CFP

CFP-Gß1+YFPGγ1

YFP-Gß1+ L-CFPGa1YFP-Gγ1+ L-CFPGa1

0

0.4

0.8

1.2

1.6

2

450 490 530 570wavelength (nm)

YFP

/CFP

L-CFPGa1+YFP-Gß1+YFP-Gγ1L-CFPGa1-Gγ1-YFPGß1L-CFPGa1-Gß1-YFPGγ1

0

0.4

0.8

1.2

1.6

2

450 490 530 570wavelength (nm)

YFP

/CFP

L-CFPGa1+YFP-Gß1+YFP-Gγ1L-CFPGa1-Gγ1-YFPGß1L-CFPGa1-Gß1-YFPGγ1

Figure 14: FRET images of CFP and YFP of Arabidopsis G protein subunits. A, Spectral encoded images of FRET between CFP and YFP of Arabidopsis G protein subunits. B, Fluorescent emission spectra from selected regions of observed cells were recorded by Zeiss LSM510 Meta spectral confocal fluorescence microscope with the excitation of chameleon multiphoton laser modulated to 820 nm.

To examine the formation of the complex among the subunits of the Arabidopsis

heterotrimeric G protein in plant cells, we co-immunoprecipitatd CFP(l)-Gα1, Gβ1, and

YFP-Gγ1 proteins transiently expressed in protoplasts. YFP-Gγ1 can be

co-immunoprecipited with CFP(l)-Gα1 using Gα1 antibody from protoplasts transfected

with a 35S-CFP(l)-Gα1-35S-Gβ1-35S-YFP-Gγ1 construct, but not from protoplasts

co-transfected with 35S-CFP and 35S-YFP-Gγ1 (Fig. 15A). CFP(l)-Gα1 and YFP-Gγ1

differe in size and can be detected together in the same gel to evaluate the efficiency of

immunoprecipitation. Thus, we conclude that the subunits of the Arabidopsis

105

heterotrimeric G protein form a complex in vivo.

The Arabidopsis Heterotrimeric G Protein Is in a ~700 kD Complex

To analyze the plasma membrane-bound complex of the heterotrimeric G protein,

we used blue native polyacrylamide gel electrophoresis (BN-PAGE) to resolve the

complex followi by the plasma membrane purification. We isolated the plasma membrane

fraction using the aqueous two-phase partitioning system (Serrano, 1984; Basboa et al.,

1987), solubilized that with NP-40 and fractionated them by BN-PAGE. BN-PAGE was

followed by a 2nd dimension denaturing SDS-PAGE gel and the proteins were detected by

Western blotting with Gα1 antibody. In plasma membrane fraction, the antibody detected

the Gα1 monomer (42 kD) in a complex of approximately 700 kD, as well as in a

low-molecular weight apparently monomeric form. Thus, Gα1 protein appeared to be part

of a complex larger than anticipated for the heterotrimer, whose molecular mass was

expected to be about 100 kD (Fig. 15B). A complex of about the same size was detected

by Western blotting with anti-CFP antibodies of a membrane fraction purified from

Arabidopsis plants expressing a CFP-Gβ1 fusion protein (Fig. 15C). However, in contrast

to what was observed with Gα1, Gβ1 protein was not observed at the expected mobility of

either the heterotrimer or the heterodimer. This observation further confirms that the

Arabidopsis heterotrimeric G protein is part of a large complex in the plasma membrane.

To determine whether the Gβ1 subunit is required for the formation of the large

complex containing the Gα1 subunit, we analyze the Gα1-containing complexes in the

106

plasma membrane fractions of agb1-2 mutant plant. Much less of the Gα1 protein is

associated with the ca. 700 kD complex in agb1-2 mutant plant than in wildtype plants, as

judged by the detection of Gα1 with anti-Gα1 on SDS-PAGE after BN-PAGE. Complexes

of several different sizes are detected with anti-Gα1 after BN-PAGE and SDS-PAGE. The

approximate sizes of the complexes are approximately 700, 400 and 150 kD (Fig. 15D).

Gα1 protein is detected in association with several smaller complexes in the 140-400 kD

range. Such Gα1-containing complexes are not observed in wildtype plants (Fig. 15B).

These observations suggest that Gβ1 participates the association of Gα1 with the large

complex, for its formation or its stability, but not for the plasma membrane localization of

Gα1. Alternatively, other proteins may bind to Gα1 in the absence of Gβ1.

Heterotrimeric G protein Signaling in the Oxidative Stress Response Requires the

Interaction of Gα1 with Gβ1

To further determine the requirement of Gβ1-Gα1 interaction for the normal function

of Gα1, we perturbed their interaction by deleting the domain from N-AtGPA6 to

N-AtGPA32 (ahGα1), which was predicted to form the first alpha-helix of Gα1 and

mediate the interaction between Gβ1 and Gα1 (Ullah et al., 2003). We kept first five

residues, which contained lipid modification sites of Gα1 and are required for the proper

localization of Gα1 (Adjobo-Hermans et al., 2006). Plants homozygous for the gpa1-4

mutations were transformed with a construct expressing Gα1 (gpa1-4 WT) or ahGα1

(gpa1-4 ahGα1) and the transformants were compared with wildtype plants for their

107

sensitivity to ozone. The gpa1-4WT plants exhibited the wildtype leaf morphology

phenotype, while the gpa1-4ahGα1 plants did not, indicating that the shortened protein can

not complement the mutation (Fig. 16A). Similarly, the wildtype construct restored the

ozone-sensitivity phenotype of mutant, while the deletion construct did not, indicating

that the mutant protein can not replace the normal protein with respect to its signaling

function in oxidative stress (Fig. 16B).

A. 1 2 3 4 5 6 7 8

B.

L-CFP-Ga

YFP-Gγ1 YFP-Gγ1

CFP

L-CFP-Ga

YFP-Gγ1 YFP-Gγ1

CFP

1 2 3 4 5 6 7 8

BN-PAGE

SD

S-P

AG

E

51 kD

Col

gpa1-4

669 440 232 140 66 kD

36 kD

BN-PAGE

SD

S-P

AG

E

51 kD

Col

gpa1-4

669 440 232 140 66 kD

36 kD

669 440 232 140 66 kD

BN-PAGE

669 440 232 140 66 kD

BN-PAGE

C.

D. 669 440 232 140 66 kD

51 kD

36 kD

BN-PAGE

669 440 232 140 66 kD

51 kD

36 kD

51 kD

36 kD

BN-PAGE

669 440 232 140 66 kD

BN-PAGE

108

A, Protein extract of protoplasts transfecting 35S-CFP and 35S-N-YFP-Gγ1 as negative control (1-4) or 35S-L-CFP-Gα1-35S-Gβ1-35S-YFP-Gγ1 (5-8) were immunoprecipited with anti-Gα1. Samples (1, 5: 10% total extract; 2, 6 concentrated flow through from the column; 3, 7: 1st elute by denaturing buffer; 4, 8: 2nd elute by denaturing buffer) were loaded in the SDS-PAGE gel and the blot was incubated with anti C/YFP antibody. B, The plasma membrane fraction from Col-0 plants and gpa1-4 mutant plants was separated in BN-PAGE gel in the 1st dimension and SDS-PAGE in the 2nd dimension. The blots were detected with Gα1 antibody. Protein mass standards were indicated by the arrow on the top of the blot. C, The total extract of agb1-2 overexpressing CFP and CFP -AtGβ1 were separated in BN-PAGE gel and the blot was detected by with anti C/YFP antibody. Protein mass standards were indicated by the arrow on the right side of the blot. D. The plasma membrane fraction from the agb1-2 was separated in BN-PAGE gel in the 1st dimension and SDS-PAGE in the 2nd dimension. The blots were detected with Gα1 antibody. Protein mass standards were indicated by the arrow on the top of the blot.

Figure 15. Detection of heterotrimeric G protein complexes in Arabidopsis

The G protein Complex Dissociates upon the Treatment of GTPγS or H2O2

To examine whether the activation of G protein causes the dissociation of Gα1 from

the complex, we used BN-PAGE to evaluate the ratio changes of complex over monomer

of plasma membrane bound Gα1 upon the treatment of GTPγS. It has been reported that

Gα subunit of rice is fully dissociated from the complex in the presence of GTPγS (Kato

et al., 2004), which binds Gα and makes it active. To test the occurrence of this reaction

in Arabidopsis, we isolated the plasma membrane fraction from wildtype plants,

incubated it with 100 μM GTPγS and separated it in BN-PAGE followed by SDS-PAGE.

We compared the ratio of complex over monomer with and without GTPγS treatment as

judged by the detection of Gα1 with anti-Gα1 on SDS-PAGE. We found that GTPγS only

caused ~30% of Gα1 to dissociate from the complex (Fig. 17A). This result is different

from that reported in rice (Kato et al., 2004). However, this result supports the

109

observation of FRET experiments performed between constitutive active Gα1 and Gβ1γ,

which shows that activation of Gα1 can not completely promote the dissociation of it

from Gβ1γ (Adjobo-Hermans et al., 2006). Taken together by those results, Gα1 subunit of

Arabidopsis may not be completely dissociated from the complex upon its activation.

To determine the direct effect of H2O2 on the activity of Gα1, we measured the GTP

binding activity of recombinant Gα1 after treatment of H2O2. The GTP binding activity of

two mammalian Gα proteins, Gαi and Gαo, can be directly regulated by reactive oxygen

species (ROS) (Nishida et al., 2000; Nishida et al., 2002; Nishida et al., 2005). To test this

possibility for Arabidopsis Gα1, we purified recombinant 6×His-Gα1 (Fig. 17C) and

assayed its GTP-binding activity before and after treatment with H2O2 (Nishida et al.,

2000; Nishida et al., 2002). However, we were unable to detect an increase in the

GTP-binding activity of recombinant Gα1 in response to H2O2 treatment (Fig. 17D, 17E),

suggesting that Arabidopsis Gα1 may not be directly activated by ROS.

To further determine whether ROS could affect G protein signaling by altering or

destabilizing G protein complex instead of directly activating it, we analyzed the ratio

complex to monomer in plasma membrane bound Gα1 upon the treatment with H2O2. We

isolated plasma membrane fractions from wildtype plants and treated them with

physiological levels of hydrogen peroxide (20 μM) prior to BN-PAGE followed by

SDS-PAGE. We found that H2O2 treatment could promote the dissociation of monomeric

Gα protein (70%) from the ~700 kD plasma membrane complex (Fig. 17B), as judged by

the detection of Gα1 with anti-Gα1 on SDS-PAGE. This observation suggests a

110

mechanism by which G protein signaling could be directly regulated by ROS and

destabilizes its complex through G protein coupled receptor or other interacting protein/s.

A. B.

0

10

20

30

40

Air

500 ppb O3

700 ppb O3

WT gpa1-4 gpa1-4wt gpa1-4ahgpa

% Io

n le

akag

e

WT gpa1-4

gpa1-4wt gpa1-4ahgpa

A, Growth phenotypes of 4 week-old light-grown plants of the indicated genetic constitution; B, Electrolyte release was assayed after a 3-hr exposure of 4 week-old plants to the indicated amount of ozone by measuring the conductivity of water in which leaves were shaken and expressed as a fraction of the total electrolytes measured.

Figure 16: Phenotypes of 4 week-old light-grown plants of the indicated genotypes constitution

A. B.

669 440 232 140 66 kD

Control

H2O2

669 440 232 140 66 kD

Control

H2O2

669 440 232 140 66 kD

Control

GTPγS

669 440 232 140 66 kD

Control

GTPγS

111

C. D.

50 KD

1 2 3 4

50 KD

1 2 3 4

600000GTP

ATP

0

200000

400000

2000 200 20 2 0

CP

MConc. (μM)

Figure 17. Redox regulation of heterotrimeric G protein complexes in Arabisopsis A and B, The isolated plasma membrane fraction from Col-0 plants was treated with buffer and 100μM GTPγS (A); 20 μM H2O2 and H2O as control (B), dissolved in the solubilization buffer and then separated in BN-PAGE gel followed by SDS-PAGE gel. The blots were detected with anti-Gα1 antibody. Protein mass standards were indicated by the arrow on the top of the blot.

C. The purification of recombinant RGS·6xHisAtGPA1 was examined in 10% SDS-PAGE gel. The lane 1 is the lysate of bacteria expressing RGS·6xHisAtGPA1 and the lane 2 is the flowthrough from the His·tag affinity beads. The lane 3 is the wash solution from the His·tag affinity beads and the lane 4 is the eluted from beads. Bench protein mark was loaded alongside of protein samples to indicate the size of the purified proteins. D, [35S]GTPγS binding to the recombinant RGS·6xHisAtGPA1 protein at different concentration of ATP and GTP. E, [35S]GTPγS binding to the recombinant RGS·6xHisAtGPA1 incubated with 20 μM H2O2 and H2O as control.

0

10000

20000

30000

0 2 5 10

Times (Mins)

CP

M

H2O2

H2O

E.

112

Discussion

We have reported that Gα and Gβγ subunits of the Arabidopsis heterotrimeric G

protein act both synergistically and separately in signaling by reactive oxygen species in

the oxidative stress response (Joo et al., 2005). Here, we provide structural and

biochemical evidence that further supports the concept that the Gα1 and Gβ1γ signals can

play both common and separate roles in varieties of development-related events and

stress-associated processes in Arabidopsis. Using FRET and immnunoprecipitation, we

provide evidence that the Arabidopsis G protein subunits interact in the plasma

membrane in plant cells. Using BN-PAGE, we make a comparison of the plasma

membrane-bound Gα1 complex between the wild type and agb1-2 mutant plant, which

suggests the concurrence of dependent and independent manner that G protein subunits

function in Arabidopsis.

Previous studies have shown that the extracellular ROS activated the

heterotrimeric G protein either directly or indirectly in the oxidative stress response (Joo

et al., 2005). Here we show that the treatment of the plasma membrane fraction with

hydrogen peroxide (H2O2) results in the partial dissociation of the Gα1 complex; however,

H2O2 can not promote the increase in GTP-binding of recombinant Gα1, which indicates

that the G protein signaling is not regulated by ROS through the direct activation of Gα1,

but likely to be mediated by some other interacting proteins in the complex.

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The Interaction of Gβ1 and Gγ1

The FRET is detected between N-terminal CFP tagged Gβ1 (CFP-Gβ1) and

N-terminal tagged YFP-Gγ1 (YFP-Gγ1) in the plasma membrane of Arabidopsis

mesophyll protoplasts, indicating the functional interaction of Gβ1 and Gγ1. This FRET

pair, as a positive control, also makes our FRET assay for all G protein subunits more

reliable. We also attempted to test the FRET in pairs of C-terminal CFP tagged Gβ1

(Gβ1-CFP) and YFP-Gγ1; CFP-Gβ1 and C-terminal YFP tagged Gγ1 (Gγ1-YFP); and

Gβ1-CFP and Gγ1-YFP. No FRET are detected in these FRET tested pairs (data not

shown), although both CFP-Gβ1 and Gβ1-CFP work as functional Gβ1 subunit with

respect to the phenotype recovery after their introduction into agb1-2 mutant plant. This

observation clearly demonstrates that the close proximity of fluorophores for FRET tested

pairs is required for FRET detection, even for the physically interacting proteins. This

result indicates that the distance between CFP-Gβ1 and YFP-Gγ1 is closer than the other

FRET tested pairs for Gβ1 and Gγ1 and proves the molecular prediction for the structure

of the Arabidopsis heterotrimeric G protein (Ullah et al., 2003), which shows that

N-terminal Gβ1 and N-terminal Gγ1 are the closest pair compared to the other

combinations.

In addition, the subcellular localization of the expressed fluorescent Gβ1 and Gγ1

was examined in Arabidopsis mesophyll protoplasts. As expected, YFP-Gγ1 is mainly

localized in the plasma membrane, where it is speculated to be anchored by its C-terminal

palmitoylated cysteine residue (Mason and Botella, 2000, 2001; Adjobo-Hermans et al.,

114

2006). Surprisingly, transiently expressed CFP-Gβ1 forms small bodies in cells and the

pattern varies from cell to cell, unlike plasma membrane localization as published before

(Obrdlik et al., 2000). Once co-expressed with YFP-Gγ1, CFP-Gβ1 is also observed in the

plasma membrane, which indicates that the plasma membrane localization of Gβ1

requires the Gγ1, although there are fewer small bodies still left in the cytoplasm, which

was reported to be localized in Golgi (Adjobo-Hermans et al., 2006). The small bodies of

CFP-Gβ1 could also be explained by the physical aggregation of transient expressing

proteins, which were not folded efficiently without enough chaperones. It has been

reported that the phosducin-like protein is an essential chaperone, required for the proper

folding of Gβ subunits in HEK-293 cells and Dictyostelium (Loew et al., 1998; Blaauw et

al., 2003; Lukov et al., 2005). Moreover, two subgroups of phosducin-like proteins have

been identified in Arabidopsis (Blaauw et al., 2003). The function of phosducin-like

proteins in Arabidopsis G protein signaling is being investigated in our laboratory.

The Interaction of Gα1 and Gβ1Gγ1

The FRET is detected between CFP(l)-Gα1 and YFP-Gγ1 with co-expression of Gβ1

in the plasma membrane of Arabidopsis mesophyll protoplasts, indicating the presence of

heterotrimeric G protein complex in Arabidopsis. However, FRET is not detected

between CFP(l)-Gα1 and YFP-Gβ1 with co-expression of Gγ1 in the plasma membrane of

Arabidopsis mesophyll protoplasts. This result is distinct from mammalian studies where

FRET can be detected between between CFP(l)-Gα and YFP-Gβ and between between

115

CFP(l)-Gα1 and YFP-Gγ (Bunemann et al., 2003). This difference may suggest the slight

structure difference between mammalian heterotrimeric G protein and Arabidopsis

heterotrimeric G protein, caused by additional residue inserts in Gα1 and Gβ1, although

the structure prediction of heterotrimeric G protein shows high conservation between

Arabidopsis and mammalians (Ullah et al., 2003). On the other hand, FRET is not

detected in all transfected cells even with CFP(l)-Gα1 and YFP-Gγ1 pair, which may

suggest that the interaction of Arabidopsis heterotrimeric G protein subunits have

transient and spatial properties like mammalian heterotrimeric G protein (Janetopoulos et

al., 2001; Ruiz-Velasco and Ikeda, 2001; Janetopoulos and Devreotes, 2002). The result

of immunoprecipitation coincides with our FRET results that not all of the YFP-Gγ1 is

co-immunoprecipitated with CFP(l)-Gα1, raising the other possibility that not all of G

protein subunits are present in the same complex.

The Complex of Arabidopsis Heterotrimeric G Protein

Indeed, BN-PAGE/SDS gel results show that some of Gα1 still remains in the same

size complex even in the absence of Gβ1, indicating some independent functions of Gα1

on Gβ1, which was shown in the roles of Gα1 in the modulation of cell division in roots

(Chen et al., 2006). This notion is also consistent with our previous report that the Gα1

protein can mediate activation of membrane-bound NADPH oxidases in the absence of

the Gβ1 protein (Joo et al., 2005). Conversely, we have also reported that a significant

fraction of the intracellular Gβ1 protein, not Gα1 protein, is ER-associated in Arabidopsis,

116

which is coincident with an independent role for the Gβ1γ complex of the heterotrimeric

G protein in cell death signaling in the unfolded protein response in Arabidopsis.

Collectively, we provide the biochemical evidence to interpret the independent function

of Gα1 and Gβ1 in numbers of physiological phenomena in Arabidopsis.

On the other hand, our mutagenesis study in Gα1 addresses the synergistic action of

Gβ1 on Gα1 because ahGα1 lacking Gβ1 interacting domain is not functional as Gα1 in the

oxidative stress response to ozone, suggesting certain signal transduction events through

Gα1 may require the entire heterotrimeric G protein complex. Although this result could

be explained by the effect of deleting the first alpha-helix on the proper folding of Gα1,

the less importance of the first 25 residues on the whole structure or folding of Gα

subunits has been formulated in the structural study of Gα subunits in mammalian cells. It

was reported that the amino-terminus of Gαt and Gαi is dynamically disordered in the

GDP bound state and becomes α-helical only upon interaction with Gβγ (Medkova et al.,

2002). Moreover, crystal structures of Gαt-GDP and Gαt-GTPγS were successfully

analyzed even without the first 25 residues (Noel et al., 1993; Lambright et al., 1994).

Thus, our results could provide another line of evidence to demonstrate the synergistic

effects of Gβ1 on some signaling event mediated by Gα1.

The Redox Regulation of Gα Complex

The dissociation of Gα from Gβγ complex upon the action by the GTP binding to

Gα has been well established in mammalian and yeast (Joo et al., 2005). In rice, this

117

phenomenon was observed (Kato et al., 2004), although in Arabidopsis this reaction is

not obviously detected by FRET and complex isolation (Adjobo-Hermans et al., 2006).

Instead, we find H2O2 promotes Gα1 to dissociate from its complex. The Gα subunit of

the heterotrimeric G protein is required for the activation of the membrane-bound

NADPH oxidase system in Arabidopsis and rice (Suharsono et al., 2002, Joo et al., 2005).

Thus, the redox regulation of G protein complex may infer a common feedback in G

protein signaling by ROS production. Despite the dissociation of Gα1 complex by H2O2

treatment, the mechanism for the dissociation of G protein complex in plants has not been

explored. Moreover, the role of signaling transduction mediated by the dissociation of G

protein complex also put an intriguing challenge for further study in plants.

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Chapter 5

CONCLUSION

Function of Heterotrimeric G Protein in Arabidopsis

Functions of heterotrimeric G protein were revealed by mutant analysis in

Arabidopsis. In this study, I also used G protein mutant: gpa1-4 lacking Gα subunit and

agb1-2 lacking Gβ subunit to analyze the functional role of heterotrimeric G protein in

Arabidopsis oxidative stress to O3 and UPR to Tm. I found that upon the O3 treatment, G

protein mutant plants have altered ozone sensitivity: gpa1-4 mutant plants are more

resitan to O3 induced tissue damages, while agb1-2 mutant plants are more sensitive to O3

induced tissue damages. This observation suggests that Gα subunit and and Gβ subunit

play different roles in cell death-associated O3 responses. To further undertand the

specific function of Gα subunit and and Gβ subunit, I monitored the expression level of

Gα subunit and and Gβ subunit in the course of O3 treatment. I found that the transcripts

of Gα subunit and and Gβ subunit were differently induced by O3, which reinforced the

concept that Gα subunit and and Gβ subunit serve different functions in O3 responses.

The oxidative burst induced by O3 was also measured in gpa1-4 mutant, agb1-2 mutant

and wildtype plants. Results show that Gα subunit is required for the production of both

peaks of the oxidative burst and Gβ subunit is only needed for the first peak of the

oxidative burst induced by O3. This observation indicates that Gα subunit and and Gβ

123

subunit can act separately in O3 responses.

Up-regulation of UPR is essential in plant defense system. Using Tm injection

method, we demonstrated that ER stress induced by Tm can cause leaf senescence in

wildtype and gpa1-4 mutant plants; however, this toxic effect was much attenuated in

agb1-2 mutant plants. This observeation indicates that independent role for the Gβ

subunit of heterotrimeric G protein in cell death signaling in the UPR. Further

measurement of induction of UPR marker gene in wildtype and agb1-2 mutant plants

show that UPR is less extensive in agb1-2 mutant than that in wildtype plants. Thus, the

Gβ subunit subunit may be directly involved in the transcriptional response and cell death

signaling in the UPR.

As in other stress responses, ROS are also implicated in the ER stress. Tm can

trigger the oxidative burst in wildtype plant, which exerts a protective role in UPR

induced cell death. Suppresing the endogenous ROS level or preventing the production of

ROS can excerabate the cell death induced by Tm. This protective effect of ROS was

confirmed by the atrbohD and atrbohD/F double mutant analysis. We find that the area of

dead tissue that develops in response to local subepidermal infiltration of tunicamycin is

much larger in ROS-inhibited plants than in untreated plants and in AtrbohD and

atrbohD/F double mutant than in wildtype plants. The protective effects of ROS are also

reflected by the fact that inhibition of ROS production by diphenylene iodonium (DPI), a

flavin oxidase inhibitor, delays the expression of UPR marker genes. Thus ROS signaling

evokes a protective response that curtails the spread of UPR-induced cell death and

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triggers the early induction of UPR gene expression.

Heterotrimeric G Protein Complex in Arabidopsis

To further investigate the underlying mechanism, I characterized the heterotrimeric

G protein complex in Arabidopsis. Using fluorescence resonance energy transfer (FRET)

and co-immunoprecipitation, I show interactions between the β and γ1 subunits and

between the α and βγ1 subunits of the Arabidopsis heterotrimeric G protein in protoplasts.

I report that both the Gα and the Gβ subunits are associated with macromolecular

complexes in plasma membrane fractions. I estimate the size of the G-protein containing

plasma membrane complex to be approximately 700 kD based on blue native

polyacrylamide gel electrophoresis, suggesting that the heterotrimeric G is complexed

with many other proteins. I find that Gα is present in both the large ca. 700 kD complex in

wildtype plants and in smaller complexes in plants homozygous for the agb1-2 Gβ null

allele. Activation of the Gα subunit with GTPγS results in partial dissociation of the Gα

subunit from the complex. Hydrogen peroxide (H2O2) promotes the extensive

dissociation of the Gα complex, but does not interfere with binding of GTPγS to purified

recombinant Gα, suggesting that reactive oxygen species affect the stability of the

complex, but not the activity of Gα itself.

125

Significance of the Present Findings

The present study has revealed two novel functions of Arabidopsis heterotrimeric G

protein in oxidative stress to O3 and ER stress to Tm. It is the first demonstration that O3

signaling perception is not from extracelluar spaces in plants but mediated by an

intracellular signaling molecule, the heterotrimeric G protein. Moreover, oxidative burst

can’t be produced by dissolution of O3 in the apoplastic fluid directly, but requires

signaling through the heterotrimeric G protein. Thus a connection between O3 signal

perception and the production of oxidative burst is established by Arabidopsis

heterotrimeric G protein.

The mechanism was further explored by which the heterotrimeric G protein

mediates ozone signaling to induce plant responses to ozone. While heterotrimeric G

protein signaling is involved in stress-associated physiological processes, information

about the structure and biochemistry of heterotrimeric G protein was not available. The

knowledge gap was filled between the structure and function of the Arabidopsis

heterotrimeric G protein. The interaction between G protein subunits using the

fluorescence resonance energy transfer technique was identified. The large complex with

other proteins of which the heterotrimeric G protein is a component was analyzed.

Surprisingly, it was found that G protein complex can be dissociated by hydrogen

peroxide treatment. This is of particular importance because this dissociation suggests

that the heterotrimeric G protein can be activated by O3-derived hydrogen peroxide

126

directly or indirectly, supporting the essential roles of G protein in plant ozone responses.

This finding significantly helps scientists to understand the mechanism of G protein

activation in the O3 stress response and to develop new strategies to derive plants with

higher O3 resistance in the long run.

Activation of UPR is not only the central part of ER stress, also critical for other

plant defense response, in which a large amount of secretory or vacuolar proteins are

induced like pathogen stress. In contrast to its extensive study in animal systems, ER

stress is an almost unexplored field in plant biology. This study links the important

signaling molecule, heterotrimeric G protein with a common biological process, ER stress

response. This signaling pathway was not even reported in animal ER stress responses

(Schroder and Kaufman, 2005). ER stress can induce cell death in plants, as it does in

animal cells. ER stress can kill cells and it is involved in a number of human diseases,

including diabetes, Gaucher’s disease, and Huntington’s disease. Given the similar toxic

effect on animal and plants, the investigation of UPR-signaling machinery in plant system

brings new insight to animal UPR study. Furthermore, it was found that the Gβ subunit of

heterotrimeric G protein is localized in the ER, which supports the notion that G protein

signaling is directly involved in ER stress.

The significance of research on the plant ER stress response lies in its connection

with other biotic and abiotic stress response. The ER is central to the cell’s ability to

coordinate its protein biosynthetic and secretory activities with calcium storage and

signaling functions. The exploration of the relationship between ER stress and oxidative

127

stress was performed. A similar oxidative burst in ER stress and oxidative stress was

demonstrated. Moreover, the protective role of the oxidative burst in UPR induced cell

death was demonstrated. Hence, the ability of plants to produce ROS in response to ER

stress curtails the spread of cell death. It has been difficult to determine whether such

ROS signaling plays a positive role in the stress response because ROS production also

contributes to cell death. However, a recent study suggests that the ability to produce

ROS curtails pathogen-induced cell death in plants homozygous for the lsd1 mutation,

which was identified through its propensity to produce “runaway cell death,” (Weymann

et al., 1995) a phenomenon in which the HR lesions observed in response to pathogens

and ROS expand to much larger sizes than observed in wildtype plants. The study reports

the paradoxical observation that cell death is even more extensive when the lsd1 null

mutation is combined with an atrbohD mutation and can be curtailed by overexpression

of the AtrbohD gene (Torres et al., 2005). These observations suggest that endogenous

ROS play a protective role, limiting the spread of cell death. The parallels between what

we observe in UPR-induced cell death and the control of HR in the pathogen response

suggests that the plant cell death response has a common physiological and molecular

basis irrespective of the causative agent. The interconnection between ER stress and

oxidative stress is of particular significance because this expands the knowledge of how

plant stress responses are interconnected.

128

Future Direction

The main question that remains to be answered in future studies is what downstream

targets of heterotrimeric G protein are. Although several Gα interacting proteins have

been identified, there is no report about Gβ protein interacting protein. Our preliminary

analysis has shown that Gβ protein is associated with a very large complex of about 700

kD, which may contain other proteins. Thus, to dissect the specific function of Gβ protein

in the ER stress, purification of Gβ-interacting protein under normal condition and ER

stress condition is needed.

Downstream targets of Gβ protein also can be hypothesized based on the

conservation of that between animal and plant system. One of the best known

downstream signaling targets of the Gβγ complex in animal cells are phospholipases

PLCβ2 and PLCβ3 (Bonacci et al., 2005; Feng et al., 2005). Moreover, accumulating

evidence shows that a plant cell’s response to pathogens and many abiotic stresses is

mediated by activation of PLC (Cessna et al., 1998; Blume et al., 2000; Andersson et al.,

2006). Hence, the activivty of PLC can be directly tested in wildtype and agb1-2 mutant

plants upon Tm treatment to determine whether PLC is the downstream target of Gβγ

complex or not under the ER stress.

It was also showed that Gβγ complex is involved in the regulation of secretory

processes in both neuronal cells and pancreatic cells (Shajahan et al., 2004; Blackmer et

al., 2005; Gerachshenko et al., 2005). In Arabidopsis, we found that Gβγ complex is

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localized in both intracellular membrane and plasma membrane. Thus, a careful analysis

of the change in subcelluar localization of the Gβγ complex in response to Tm may also

provide insight of its function in UPR.

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References

Andersson, M.X., Kourtchenko, O., Dangl, J.L., Mackey, D., and Ellerstrom, M. (2006). Phospholipase-dependent signalling during the AvrRpm1- and AvrRpt2-induced disease resistance responses in Arabidopsis thaliana. Plant J 47, 947-959.

Blackmer, T., Larsen, E.C., Bartleson, C., Kowalchyk, J.A., Yoon, E.J., Preininger, A.M., Alford, S., Hamm, H.E., and Martin, T.F. (2005). G protein betagamma directly regulates SNARE protein fusion machinery for secretory granule exocytosis. Nature Neuroscience 8, 421-425.

Blume, B., Nurnberger, T., Nass, N., and Scheel, D. (2000). Receptor-mediated increase in cytoplasmic free calcium required for activation of pathogen defense in parsley. Plant Cell 12, 1425-1440.

Bonacci, T.M., Ghosh, M., Malik, S., and Smrcka, A.V. (2005). Regulatory interactions between the amino terminus of G-protein betagamma subunits and the catalytic domain of phospholipase Cbeta2. The Journal of Biological Chemistry 280, 10174-10181.

Cessna, S.G., Chandra, S., and Low, P.S. (1998). Hypo-osmotic shock of tobacco cells stimulates Ca2+ fluxes deriving first from external and then internal Ca2+ stores. The Journal of Biological Chemistry 273, 27286-27291.

Feng, J., Roberts, M.F., Drin, G., and Scarlata, S. (2005). Dissection of the steps of phospholipase C beta 2 activity that are enhanced by G beta gamma subunits. Biochemistry 44, 2577-2584.

Gerachshenko, T., Blackmer, T., Yoon, E.J., Bartleson, C., Hamm, H.E., and Alford, S. (2005). Gbetagamma acts at the C terminus of SNAP-25 to mediate presynaptic inhibition. Nature Neuroscience 8, 597-605.

Schroder, M., and Kaufman, R.J. (2005). ER stress and the unfolded protein response. Mutation Research 569, 29-63.

Shajahan, A.N., Tiruppathi, C., Smrcka, A.V., Malik, A.B., and Minshall, R.D. (2004). Gbetagamma activation of Src induces caveolae-mediated endocytosis in endothelial cells. The Journal of Biological Chemistry 279, 48055-48062.

Torres, M.A., Jones, J.D., and Dangl, J.L. (2005). Pathogen-induced, NADPH oxidase-derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana. Nature Genetics 37, 1130-1134.

Weymann, K., Hunt, M., Uknes, S., Neuenschwander, U., Lawton, K., Steiner, H.Y., and Ryals, J. (1995). Suppression and Restoration of Lesion Formation in Arabidopsis lsd Mutants. Plant Cell 7, 2013-2022.

Weymann, K., Hunt, M., Uknes, S., Neuenschwander, U., Lawton, K., Steiner, H.Y., and Ryals, J. (1995). Suppression and Restoration of Lesion Formation in Arabidopsis lsd Mutants. Plant Cell 7, 2013-2022.

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Curriculum Vitae

Shiyu Wang

Education Aug. 2001 to Aug. 2007 Ph. D

Biochemistry, Microbiology and Molecular Biology Department, Penn State University, U.S.A. Sep. 1998 to Jun. 2001 M. S.

Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, China Sep. 1994 to Jun. 1998 B. S.

Biochemistry and Molecular Biology Department, Jilin University, China

Publication:

Shiyu Wang, Yan Feng et al, Archives of Biochemistry and Biophysics 411 (2003), 56–62; Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii.

Shiyu Wang, Savitha Narendra, and Nina Fedoroff, Proceedings of the National Academy of Sciences of United States of America 104 (2007), 3817-3822; Heterotrimeric G protein signaling in the Arabidopsis unfolded protein response.

Junghee H. Joo, Shiyu Wang, J.G. Chen, A.M. Jones, and Nina V. Fedoroff, the Plant Cell 17 (2005), 957–970; Different signaling and sell death roles of heterotrimeric G protein a and b Subunits in the Arabidopsis oxidative stress response to ozone.

Yanru Wang, Gao Gui, Shiyu Wang, Zhi Wang, Yan Feng, Act Scientiarum

Naturalium Universitiis Ilinenesis 10 (1999), 91-95; Selection of the lipase-producing strains and some properties of partial purified lipase. Awards

Homer F. Braddock Fellowship and Nellie H. and Oscar L. Roberts Fellowship for outstanding students in Penn State Unviersity