Arabidopsis Response to the Carcinogen Benzo[A]Pyrene
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ARABIDOPSIS RESPONSE TO THE CARCINOGEN BENZO[A]PYRENE
A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAW AIT IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
TROPICAL PLANT AND SOE. SCIENCES
DECEMBER 2008
By Beth Irikura
Dissertation Committee: Robert Pauli, Chairperson Henrik Albert Paul Moore Ming-Li Wang Paul Patek We certify that we have read this dissertation and that, in our opinion, it is satisfactory in scope and quality as a dissertation for the degree of Doctor of Philosophy in Tropical Plant and Soil Sciences.
DISSERTATION COMMITTEE
Chairperson
11 ©Copyright by Beth Irikxira 2008 All Rights Reserved
111 ACKNOWLEDGEMENTS
I am fortunate to have been able to design a project that has held my interest throughout. Funding was provided in part by ARCS Foundation awards and a U.S. Dept, of Education GAANN Fellowship in Interdisciplinary Biotechnology. I’d like to acknowledge the people and situations that made this research possible. First, I thank my father for demonstrating a profound love of science, while encouraging his children to explore all other conceivable options.
My mother’s death by lung cancer at the age of 54 interrupted my college education, too late to stop me from getting a BA in English literature, but so early that it forced me to reevaluate priorities. My mother also pioneered in our family, showing that an artist could teach science. The last photo of my mother is from late 1983. She’s dressed up as a chicken, and she’s laughing. I like to think she was trying to signal the connection between avian sarcoma virus and cancer, which provided the first molecular understanding of cancer processes. At the time, that connection was just beginning to be deciphered, and public understanding was at the level of thinking maybe you shouldn’t eat too much chicken.
It’s hard to explain how I came to work with BaP, a chemical that very likely contributed to my mother’s death. My first project measured BaP interactions with monoclonal antibodies that had been produced by inducing cancers in mice. Yet without that initial work, it’s unlikely that I would have proposed this project. The ensuing years allowed me to develop as a scientist, and more importantly allowed the field to make major progress that I could later use. The situations and people that have impeded my progress (data not shown) have left some lasting scars, but the delay has meant that many exciting advances have been made in the meantime, which give my data a richer and more meaningful context. Without time served, this dissertation would be (even) more like an interpretive dance.
Heartfelt thanks to my current advisor and committee members for their participation, insight, and support. Research is only possible through the sacrifices of people wiser and more patient than us.
It is impossible and possibly impolitic to thank everyone who has helped, encouraged, or inspired me, but I am enormously grateful to all the people I’ve had the pleasure to work for and with. You know who you are; please accept my thanks. I thank my family for being beautiful, patient and there. To Kawai, Dasmin, Jonah, Joe, Kaimanu, Chase, Pele, Marley, Azzie and Ka‘eo: I’m sorry for working so much, I love you, go to college.
Finally, this is for Aike, who was always there.
IV ABSTRACT
This project investigates the effects of the carcinogenic environmental pollutant benzo[a]pyrene (BaP) on the model plant, Arabidopsis thaliana ecotype Columbia.
Previous research has demonstrated phytodegradation of BaP, in the presence and absence of microorganisms. BaP and its metabolites have been detected in plant tissues, but the parent compound is only found in parts-per-billion quantities in most plants.'
Increases in plant biomass and lifespan have been observed after growth in BaP, raising questions about how plants are able to benefit from a compound that is detrimental to most other eukaryotes. Since plants share many of the same molecules used by animals in response to BaP (e.g. cytochrome P450, peroxidases, laccases, glutathione, glycosylases and aminotransferases), it has been assumed that they employ similar mechanisms to degrade BaP. Until now, investigations of these mechanisms were limited by methodological constraints. In this study, we applied genetic and genomic tools to
determine specific gene expression responses to BaP in Arabidopsis. Particular attention
was paid to the mechanisms by which BaP tolerance was achieved in the plant. Much
research has shown functional equivalence between homologous genes in plants and
animals, hence the results of this study may have applications in biomedical research on
molecular mechanisms of BaP effects in mammals. TABLE OF CONTENTS
page
Acknowledgements...... iv
Abstract...... v
Table of Contents...... vi
List of Tables...... xii
List of Figures...... xiii
List of Abbreviations...... xv
Chapter 1. Introduction...... 1
Chapter 2. Background and Literature Review
2.1 Benzo[a]pyrene as a model carcinogen
2.1.1 PAHsandBaP...... 3
2.1.2 Benzo[a]pyrene metabolism...... 3
2.1.3 BPDE in cancer...... 4
2.1.4 BaP quinones, hydroxy-BaP, and oxidative metabolites...... 4
2.1.5 BaP as an endocrine dismptor...... 5
2.1.6 BaP alters gene expression in multiple, interacting pathways...... 6
2.2 Phytoremediation
2.2.1 Plants can remediate BaP...... 7
2.2.2 Routes of exposure...... 10
2.2.3 Selection of plant...... 12
2.3 Expression Analysis
2.3.1 Microarrays...... 13
vi 2.3.2 Quantitative RT-PCR...... 14
2.3.3 Expression profiling...... 15
2.3.4 Bioinformatics...... 16
2.3.5 Gene expression profiling identifies additional genes and pathways 18
2.4 Related plant research
2.4.1 Plant response to PAH and other xenobiotics...... 19
2.4.2 Agrobacterium-indMCQd tamors—a plant cancer model?...... 21
2.5 Cross-kingdom comparisons...... 22
Chapter 3. Hypotheses and Objectives
3.1 Hypotheses...... 26
3.2 Objectives...... 26
Chapter 4. Plant Uptake of BaP and Observable Changes
4.1 Introduction...... 27
4.2 Methods
4.2.1 Growth conditions and phenotypic measurements...... 28
4.2.2 Microscopic evidence of BaP effects...... 30
4.2.3 HPLC to detect reduction of BaP in agar...... 30
4.2.4 Comet assay to measure DNA damage after root exposure to BaP...... 30
4.3 Results
4.3.1 Phenotypic changes in Arabidopsis following exposure to BaP...... 32
4.3.2 Microscopic evidence of BaP effects...... 33
4.3.3 HPLC to detect reduction of BaP in planted agar...... 34
4.3.4 DNA damage detected by the comet assay...... 34
vii 4.4 Discussion...... 43
Chapter 5. BaP Alters Gene Expression of Plants Grown in BaP for 4 Weeks
5.1 Introduction...... 48
5.2 Methods
5.2.1 Plant growth conditions in agar...... 51
5.2.2 Affymetrix ATHl GeneChip analysis of agar-grown plants...... 51
5.2.3 qRT-PCR of plants grown in soil...... 52
5.2.4 Bioinformatics and data mining...... 56
5.3 Results
5.3.1 Phenotypic observations...... 58
5.3.2 Microarray expression results...... 58
5.3.3 Variation among the same class of genes...... 59
5.3.4 Quantitative RT-PCR results...... 59
5.4 Discussion
D O X l...... 73
Lac7...... 74
Prx3a...... 75
MRP3...... 76
GH17...... 77
AtNAP...... 77
NUDT7...... 79
MYB4...... 80
SS...... 81
viii Chapter 6. Expression Analysis of Plants Grown in BaP for 24 hours
6.1 Introduction...... 84
6.2 Methods
6.2.1 Plant growth conditions in sand...... 85
6.2.2 Affymetrix ATHl GeneChip analysis of plants grown in sand...... 86
6.2.3 Repeat of experiment in so il...... 86
6.2.4 Quantitative RT-PCR of plants grown in soil...... 87
6.2.5 Data analysis and bioinformatics...... 88
6.3 Results
6.3.1 General observations...... 88
6.3.2 Microarray analysis of 24 h gene expression in sand-grown plants...... 89
6.3.3 Gene expression changes after 24 h in soil-grown plants by qRT-PCR ....89
6.3.4 Comparison of qRT-PCR and microarray results...... 89
6.3.5 Promoter analysis of genes identified by microarray...... 89
6.3.6 Intersection of BaP and cold stress response...... 90
6.4 Discussion...... 99
Hormones...... 102
P450...... 105
A B C & U G T l...... 105
GH17...... 106
A tl4a...... 107
E4&E5...... 107
FK Fl...... 108
ix TO Cl...... 108
GRP7...... 109
RD29A...... 110
BoCAR...... 110
Small hydrophobic protein...... I l l
VAMP713...... I l l
W A K l...... 112
Jmj...... 113
BT4...... 113
HSP70...... 114
Chapter 7. Summary and Future Directions
7.1 Concluding remarks
a. Cross-kingdom comparisons...... 117
b. Viruses and BaP...... 118
c. Benefits of BaP?...... 119
d. Plant-specific responses...... 119
e. Microarrays identify cancer pathways...... 121
7.2 Obstacles and future directions
a. Comet assay...... 122
b. SA& NSAIDs...... 123
c. Protein assays...... 124
d. Proteomic profiling...... 124
e. Detecting transcripts after treatment with a mutagen...... 125
X f. Phytoremediation and cancer...... 126
7.3 References...... 153
7.4 Appendices
Appendix A. Mammalian genes up-regulated by BaP or BPDE...... 128
Appendix B. Mammalian genes down-regulated by BaP or BPDE...... 133
Appendix C. Complete gene list for 4-wk exposure microarray results...... 138
Appendix D. Complete gene list for 24-hour exposure microarray results...... 146
Appendix E. Primers used for qRT- PCR...... 151
XI LIST OF TABLES
Table 4.1 T-test for phenotypic differences between control plants and plants grown in 40 ppm BaP...... 33
Table 5.1 Differences in the experimental conditions in the two 4-wk growth experiments...... 62
Table 5.2 Genes called Increased or Decreased in 4-wk GeneChip results...... 63
Table 6.1 Twenty-eight genes represented by 25 probe sets that increased in the 24-h microarray results...... 92
Table 6.2 Twenty-nine genes represented by 25 probe sets that decreased in the 24-h microarray results...... 93
Table 6.3 Analysis of promoter motifs at 24 h and 4 wks...... 94
Table 6.4 BaP-upregulated genes containing DREs in 1 kb upstream are regulated differently by other abiotic stresses...... 95
X ll LIST OF FIGURES
Figure 2.1 Phase I activation of BaP in animals (A) and diagram illustrating AhR- dependent effects of BaP (B)...... 24
Figure 4,1 Stratified seeds germinated faster on Difco Bacto agar with 50 ppm BaP in 0.2% DMSO than on agar with DMSO alone...... 35
Figure 4.2 Comparison of fluorescent bodies in root tissue...... 36
Figure 4.3 Osmium tetraoxide stained roots suggest higher levels of phenolics and/or lipophilic metabolites in BaP-grown plants...... 37
Figure 4.4 HPLC analysis of agar shows a decrease in BaP when plants are present.. .38
Figure 4.5 Darkening of media and roots in the presence of BaP...... 39
Figure 4.6 Types of nuclei observed in alkaline comet assay...... 40
Figure 4.7 DNA damage measured by comet assay...... 41
Figure 4.8 Comet assay of sand-grown Arabidopsis shows individual differences in DNA damage within 24 h of exposure to BaP...... 42
Figure 5.1 Effects of methylobacteria on BaP-grown and control plants...... 66
Figure 5.2 No clear phenotypic differences between soil-grown control plants and BaP-grown plants...... 67
Figure 5.3 Comparative distribution of cellular components based on Gene Ontology Annotation...... 68
Figure 5.4 Differential GO Annotation of Biological Processes in control V5. BaP- exposed plants...... 69
Figure 5.5 Differential GO Annotation of Molecular Function in control vs. BaP- exposed plants...... 70
Figure 5,6 Expression of 10 genes in soil-grown Arabidopsis, by qRT-PCR...... 71
Figure 5.7 Comparative expression of 10 genes measured in two independent experiments, expressed as the ratio of BaP vs. control plants...... 72
Figure 6.1 Specific gene expression in 24 h soil-grown plants by qRT-PCR...... 96
xiii Figure 6.2 Comparison of GeneChip and qRT-PCR results for sand- and soil-grown plants, respectively...... 97
Figure 6.3 Microarray results for 24-h BaP overlaid on map of abiotic stresses mediated by two DRE-binding transcription factors...... 98
XIV ABBREVIATIONS a. a. Amino acids
ABA Abscisic acid
ABC ATP-Binding Cassette (ABC) superfamily; used here for At3g59140
ABRC Arabidopsis Biological Resource Center
AhR Aryl hydrocarbon receptor
Atl4a At3g28300, shares probe set with At3g28290, contains EOF repeats
AtNAP At4g04460 Napsin homolog
BaP Benzo[a]pyrene
BCAT3 Branched chain aminotransferase 3, AT3G49680
BCF Bioconcentration factor, used here for PAH concentration in fresh parts of
plant/p AH concentration in dry soil
BLASTN Basic Local Alignment Search Tool for nucleotide comparison
BoCAR B. oleracea Controlled Atmosphere Responsive 6-4 homolog; Atlgl7665
BPDE Benzo[a]pyrene diol epoxide
BPQ Benzo[a]pyrene quinone(s)
BT4 At5g67480, Transcription Adaptor putative Zinc finger and BTB
domain-containing protein 4 cDNA Complementary DNA
CYP Cytochrome P450
dCt Delta Ct, difference in threshold crossing points in qRT-PCR
DOXl At3g01420, a-dioxygenase 1, also called PIOX
XV E Expect value; low E reflects low probability of finding a particular
comparison score by chance
El Used here for expressed protein Atlgl3650, of unknown function
E4 Used here for expressed protein At4g33980, of unknown function
E5 Used here for expressed protein At5g42900, of unknown function
EE Evening Element, AAAATATCT promoter recognition site
EGF Epidermal Growth Factor
ER Stress Conditions disrupting normal endoplasmic reticulum function, associated
with unfolded protein response (UPR)
ERE Ethylene Response Element, recognition site within gene promoters
EREBP/ERF ERE-Binding Protein, also called Ethylene Response Factor
EROD Ethoxyresorufin-O-deethylase, assay used to measure CYPl A1 activity
FGFl Fibroblast growth factor 1, mammalian mitogen
FICZ 6-formylindolo[3,2-b]carbazole
FITC Fluorescein isothiocyanate
FKFl Flavin-binding kelch repeat F box /Atlg68050, also known as Adagio 3
GA Gibberellic acid gDNA Genomic deoxyribonucleic acid
GFG H. sapiens FGF-Antisense gene, also called Nudt-6
GMBF Greenwood Molecular Biology Facility at University of Hawaii
HomoloGene Gene homolog identified in NCBI HomoloGene database, available at:
http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene
hydrophobic Used here to refer to small hydrophobic protein At4g30650
XVI HPLC High performance liquid chromatography
HSP Heat shock protein lAA Indole acetic acid, auxin
JA Jasmonic acid
Jmj Jumonji C-domain containing protein, used here for At3g20810
Kow Octanol-water coefficient at equilibrium at a specific temperature
LAC7 Laccase At3g09220
MBBE Molecular Biosciences & Bioengineering Department at Univ. of Hawaii
MeJA Methyl jasmonate
MIPS Munich Information Center for Protein Sequences
MMS Methyl methanesulfonate miRNA MicroRNA, about 21-23 nucleotides long, single-stranded, binds
complementary mRNA sequences, causing silencing mRNA Messenger RNA
MRP3 At3gl3080, multidrug resistance protein 3
MS Murashige-Skoog macro- and micronutrients with Gamborg’s B5 vitamins
NCBI National Center for Biotechnology Information
nt Nucleotides
NUDT7 NUDIX hydrolase (nucleoside diphosphates linked to moiety X)-type
protein, At4gl2720, homolog for human GFG
P450 Generic term for CYP450, used here for At3g28740
PAC Motif C-terminal to Per-Amt-Sim motif, may contribute to PAS fold
PAH Polycyclic aromatic hydrocarbon
xvii PAGE Polyacrylamide gel electrophoresis
PAS Per-Amt-Sim domain
PCR Polymerase Chain Reaction ppb Parts per billion, equivalent to pg/kg or pg/L ppm Parts per million, equivalent to mg/kg
PRX3a Peroxidase At5g64100 qRT-PCR Quantitative reverse transcription PCR
RD29A At5g52310, major regulator of cold, ABA, and drought stress response
RT-PCR Reverse transcription PCR
SA Salicylic acid
SEM Standard error of the mean siRNA Small interfering RNA, about 20-25 nt long, double-stranded
STRS Strictosidine synthase, used here for Atlg74010
TAE Tris-acetate-ethylenediamine tetraacetic acid buffer
TBE Tris-borate-ethylenediamine tetraacetic acid buffer
TAIR The Arabidopsis Information Resource
TF Transcription Factor
TIGR The Institute for Genomic Research
UGTl Atlg05560, homolog of conserved UDP GlycosylTransferase
UV Ultraviolet
Vamp713 Vesicle-Associated Membrane Protein 713, At5gl 1150
WAKl Wall-Associated Kinase 1, Atlg21250
XRE Xenobiotic Response Element, consensus binding site=TNGCGTG
xviii CHAPTER 1 Introduction
1,1 Project overview
This project investigated the potential for phytoremediation of benzo[a]pyrene
(BaP) and the mechanisms by which plants are able to tolerate or degrade BaP. Previous studies have shown that this mutagen affects plants differently than animals.Given the extensive homology between eukaryotic genomes, a comparison of plant and animal global gene expression might provide insight into how these responses work.
Preliminary experiments investigated different BaP concentrations, solvents, and growth media. Phenotypic observations were used to explore the potential of Arabidopsis to take up, tolerate, and degrade BaP. The alkaline comet assay confirmed BaP uptake by shoots and indicated deleterious effects of BaP on shoot DNA. Affymetrix Arabidopsis ATHl
GeneChip microarrays were used to identify alterations in gene transcription in 4-wk old plant shoots grown in a high but sublethal dose of BaP (50 ppm). Three biological replicates were processed and analyzed statistically at two exposure time points.
Bioinformatics databases and software were used to link the up- and down-regulated genes to biochemical pathways. To ensure reproducibility and to extrapolate the results to environmental exposures, growth experiments were repeated, with plants grown in potting soil instead of sterile agar or sand. Expression profiles of representative genes at different exposure timepoints were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) on soil-grown plants to confirm significance and to look for time- and condition-dependent responses. By combining two well-studied models (plant and chemical), new information about both Arabidopsis and BaP was
1 found. Potential genetic candidates for phytoremediation and biochemical studies of the effects of BaP were identified. CHAPTER 2 Background and Literature Review
2.1 Benzo[a]pyrene, a model carcinogen
2.1.1 PAHs and BaP
Polycyclic aromatic hydrocarbons (PAHs) are among the most common and persistent environmental pollutants. They are produced by incomplete combustion arising from anthropogenic and geological processes. Benzo[a]pyrene (BaP) has five aromatic rings, and is among the most dangerous of the PAHs. It is a major toxicant in cigarette smoke, charred food, coal tar, diesel exhaust, and volcanic eruptions. BaP’s extreme hydrophobicity facilitates its binding to soil and airborne particles, enabling it to persist in the environment. BaP causes cancer in mammals, and is often used to study carcinogenesis in cell lines or animals.
2.1.2 Benzo[a]pyrene metabolism
BaP metabolism is generally understood as a three step process. BaP activation by phase I enzymes can be complemented by phase II enzymes which tend to make the
compound more hydrophilic (i.e., glutathione S-transferase adding glutathione) and
facilitate its breakdown and elimination by phase III enzymes (such as ABC
transporters)."^ Figure 2.1 illustrates phase I activation pathways for BaP. In animals,
activation begins with BaP binding to the aryl hydrocarbon receptor (AhR) in a complex
with two 90 kDa heat shock proteins and the aryl hydrocarbon receptor-interacting
protein AIP.^ BaP-bound AhR changes conformation, dissociates from that complex and
translocates to the nucleus, where it complexes with Arat (AhR-nuclear translocator) and
affects transcription of genes containing the xenobiotic responsive element, XRE (5’- TNGCGTG-3’).^ .The most mutagenic metabolite of BaP derives from this pathway, via
AhR-Amt mediated induction of cytochrome P450 lAl. CYP450 transforms the relatively less reactive BaP into electrophilic benzo[a]pyrene diol epoxide (BPDE). This form of BaP is especially adept at binding DNA, and usually adducts to deoxyguanosine or deoxyadenosine nucleotides (G>A).’ As mutations accumulate and affect critical genes for cell cycle control, detection and repair of DNA damage, the cell moves closer to a cancer phenotype.
2.1.3 BPDE in cancer
Mutation via BPDE adduction to specific nucleotides within tumor suppressor protein P53 gene has been implicated in many forms of cancer, particularly those of epithelial origin.*’® There is an especially high correlation with lung cancer. Not only have BPDE-induced lesions been traced to a particular gene (tumor suppressor P53), but nucleotide preferences have been demonstrated. BPDE-induced mutations that lead to cancer occur primarily at dG, usually at methylated CpG sites, and mostly at specific codons within P53 (157, 248, and 273).^° In human cells, BPDE adducts reached a maximum at one hour following exposure." DNA repair processes eliminated most adducts, but 40-45% remained after 23-48 hours in this study.
2.1.4 BaP quinones, hydroxy-BaP, and oxidative metabolites
CYP450 and other Phase I enzymes have been shown to transform BaP into quinones and mono- or di-hydroxy forms that can also bind DNA, or cause oxidative damage." Recent evidence indicates that BaP quinones (BPQ) have mutagenic and
carcinogenic potential similar to that of BPDE." BaP quinone metabolites result from plant and algal, as well as animal and fungal degradation." Enzymatic degradation of
4 BaP generates toxic free radicals, and the enzymes induced by BaP can also act on endogenous substrates to produce damaging compounds. For example, BaP-induced cyclooxygenase 2 (also known as prostaglandin endoperoxide synthase, or COX-2) can oxidize BaP, but also acts on lipids to produce prostaglandins and the toxic by-product malondialdehyde (MDA), and both oxidized BaP and MDA can form adducts with deoxyguanosine or deoxyadenosine.'^ Mammalian aldehyde dehydrogenases, which can break down these endogenous aldehydes, are induced by BaP exposure,'^ and ALDH class 3 enzymes are up-regulated in hepatoma cells.'’ Strolin Benedetti, et al. (2006) suggest that the focus on CYP450 assays for BaP oxidation may have led to an underestimation of the roles that non-CYP450 enzymes play in BaP response.'*
Mammalian flavin-containing monooxygenases (FMOs) are broad-specificity enzymes
that oxidize xenobiotics like BaP. In plants, FMOs catalyze reactions in auxin synthesis
and glucosinolate metabolism, and mediate pathogen response,'^ but could conceivably
act on BaP as well.
2.1.5 BaP as an endocrine disruptor BaP and its metabolites have steroid-like structures, and exhibit endocrine
disrupting activity.’" BaP and BPDE have been found to lower the levels of constitutive
and testosterone-induced androgen receptors in several human cell lines.’' The
metabolism of prolactin and growth hormones was increased by BaP concomitantly with
induction of CYP450.” Estrogen and estrogen mimics are implicated in breast cancer
and other cancers, and BaP, 3-hydroxy BaP and 9-hydroxy BaP have been shown to bind
the estrogen receptor.” BaP was also shown to increase production of carcinogenic and
genotoxic oxygenated forms of estradiol, suggesting a possible mechanism for estrogen potentiation of cancer risk.^"* BaP metabolites also act on signaling pathways related to
hormone response. The tumor suppressor protein, BRCA-1, is produced at low levels in
breast and ovarian cancers as well as in BaP- or BPDE-exposed cells; therefore the
interaction between this gene, BPDE, and estrogen receptors may have significance for
carcinogenesis. Estrogen activation of BRCA-1 requires promoter binding by the
estrogen receptor and unliganded AhR. BPDE represses transcription, apparently by
interfering with AhR binding to the promoter.^^’^^’^’
The planar aromatic ring structure of BaP resembles the structures of natural and
synthetic plant hormones, especially 1-naphthalene acetic acid. Hormone signaling
disruption by BaP is suggested by experiments in which the levels of auxin and cytokinin
were altered after fern gametophytes were exposed to BaP.^* An earlier study had also
found that lower BaP concentrations stimulated growth and auxin levels, while higher
concentrations decreased ‘extractable auxin’ and antagonized fern growth.^^ Thus it
seems that BaP may act as a hormone mimic in both animals and plants. The
mechanisms behind auxin’s dominant role in plant development involve interactions
between the F-box TIRl auxin receptor and SCF ubiquitin ligase-associated proteasomal
degradation.The proteasome is implicated in cancer progression,^' so the identification
of direct proteasome regulation by a small molecule suggests additional mechanisms by
which BaP may perturb cellular processes and contribute to malignancy.
2.1.6 BaP alters gene expression in multiple, interacting pathways
The interactions of BaP with individual genes occur in the context of complex,
interlocking pathways. The chemotherapy target cyclooxygenase 2 {COX-2) is up-
regulated in many cancers and contributes to inflammation as well as activation of BaP.
6 COX-2 induction by BPDE is implicated in bone cancer, and appears to require estrogen receptor, phosphoinositol 3 kinase/ downstream effector Akt (PI3K/Akt) and extracellular
signal receptor kinase/mitogen activated protein kinase (ERK/MAPK), as chemical repressors of these pathways decreased COX-2 expression and osteoblast proliferation.^^
In esophageal cancer lines, up-regulation of COX-2 is preceded by down-regulation of
retinoic acid receptor beta {RAR-ji) by BPDE.^^ BPDE-induced methylation of CpG sites
in the promoter of RAR-fi results in loss of expression and occurs in many tobacco-related
cancers.^'*’^^’^^ By tracing changes in gene expression to an epigenetic modification,
cytosine methyltransferase was identified as a target for chemotherapeutic
intervention.^’’^*
2.2 Phytoremediation
2.2.1 Plants can remediate BaP
BaP can be efficiently degraded by bacteria and fungi (bioremediation), but some of
the most interesting results have been obtained with plants. Early phytoremediation
studies sought to replicate environmental scenarios, employing an often unidentified
consortium of microbes, plants, and even small soil animals.In these situations it
initially seemed that the microorganisms were responsible for the bulk of the degradation,
especially when they continued to perform well after isolation and in vitro culture.
However, a substantial increase in BaP degradation in planted versus unplanted soils has
been demonstrated."^' Studies have shown that the addition of crushed plant tissues or
extracts can replicate at least some of the effects of the living plant."'’’"'* Translocation of PAHs from soil to soybean seeds was observed in field tests, and the authors concluded that translocation in the xylem occurred."^
Most importantly, plants have been shown to take up and degrade BaP in sterile conditions/^ There is a report of plants reducing radiolabeled BaP to carbonic acid and related intermediate compounds.'*® One research group found that soybean and
Chenopodium rubrum cell cultures transformed approximately 70% of added radiolabeled BaP into a combination of soluble metabolites and insoluble bound residues,'*’’'*^ but this contradicts the general pattern wherein PAHs with three aromatic rings are more easily degraded than five-ring PAHs.'*^ A later study challenged these results. Working with the four-ring pyrene, Hiickelhoven, et al. (1997) found that only
6.4% of pyrene was transformable by soybean cell suspension cultures, and only into bound residues. Different species were able to transform pyrene more efficiently: purple foxglove callus cultures transformed almost 38% of pyrene, and wheat suspension cultures transformed up to 94%. The authors also found that incorporation into bound residues seemed to be the main fate of pyrene, and identified the most abundant metabolite as a 1-hydroxypyrene methyl ether, confirming theories that O- methyltransferases could act on PAHs as well as native phenolics.®”’®’
Other studies, however, dispute the idea that plants contribute significantly to the degradation. Rooted poplar cuttings produced only a very low concentration of methoxy-
BaP, and the authors questioned whether it could be attributed to active metabolism by the plants.®^ In Plantago lanceolata, only root uptake could be demonstrated, with no evidence of translocation of radiolabeled BaP or metabolites to the aerial portions.®^
Probably the best theory for plant uptake of BaP is that adhesion to the root stimulates
8 enzyme and/or exudate secretion into the rhizosphere, making BaP more soluble and facilitating uptake. The majority of current research continues to focus on rhizosecretion of factors stimulating microbial bioremediation of soil PAHs.
Nevertheless, BaP concentrations decrease in media in which plants are growing, and BaP has been reported to have various effects on plants. Not all of the effects are negative, although high concentrations can be toxic to some plants. In a study using fern gametophytes, moderate doses (3.2 pg/mL) inhibited growth, and higher doses (10 pg/mL) decreased spore germination and plant survival.^"' In contrast, lower doses (0.1 to
0.32 pg/mL) accelerated the onset of morphogenesis. Legumes grown in BaP showed decreased root and shoot biomass, and inhibition of mycorrhizal associations.^^ Most interestingly, BaP has been found to have a growth stimulating effect on fems^"* and hemp plants,^® and it extended the lifespan of Arabidopsis in this study. This contrasts substantially with its effects on animals.
In mammals, the first steps of BaP metabolism (oxygenation by CYP450, cyclooxygenases, and epoxide hydrolases) produce more reactive compounds that can lead to cancer. Carcinogenesis involves an abnormal increase in cellular proliferation and suppression of apoptosis. Either plants may be able to metabolize BaP more completely, or they may be able to regulate proliferation and apoptosis in ways that promote survival. The difference in outcome may be due to plants lacking particular genes or pathways, such as tumor suppressor P53, or because plants possess unique genes which enable protective biochemical responses. However, since genome sequencing has revealed the existence of plant homologs of many genes known to be involved in cancer initiation and promotion, there may be more similarities than are at first evident. For
9 these reasons, and to maximize the potential applications of phytoremediation, it seemed valuable to investigate the molecular mechanisms underlying plant tolerance to BaP.
2.2.2 Routes of Exposure
Studies of PAH content in plant material have indicated that most of the low molecular weight PAHs (containing two or three fused aromatic rings) found in aerial portions of plants, including seeds, are probably a result of atmospheric deposition rather than uptake through roots.It appears also that the smaller PAHs may volatilize out of plant tissue when exposed to higher temperatures.^^ For higher molecular weight PAHs, root uptake has been shown to be more common and Fismes et al. (2002) conclude that this is the main route of uptake for PAHs with five or more aromatic rings. BaP, with a log Kow of approximately 6, is hydrophobic enough to diffuse passively through membranes to the cytoplasm, where it can be oxidized and conjugated to sugar or glutathione molecules. Compounds with a log Kow between 0.5 and 3 are more easily transported in the xylem, so more extensively modified BaP is more likely than unmodified BaP to be translocated in the shoot.®*’ Oxidation and conjugation products are both more soluble and more likely to be actively transported to the vacuole, where they may be sequestered or further degraded.®’ Root uptake of two 3-ring PAHs was visualized by two-photon excitation microscopy.®^ In a survey of PAH concentrations in vegetables, researchers found low bioconcentration factors (BCF) for 5- and 6-ring PAHs
in potato and carrot leaves (0.01 e-2), and a BCF of 0.01 e-4 to 0.04 e-4 in the root pulp,
compared with 27-327 ppm in soil.®^ In another study, BaP content in dry grass shoot
tissue was measured at 3-23 ppb, and 8-390 ppb in dry roots.®^ Other researchers report
being unable to measure BaP concentrations in plants used for high concentration
10 phytoremediation experiments (including exposure to more than 20 ppm BaP), because
levels in plant tissues were below the HPLC limit of detection.^"* Similar analysis
constraints and the small plant size of Arabidopsis rendered measurement of BaP
concentrations in this diminutive laboratory plant not feasible with available technology.
When researchers have been able to measure BaP in plant tissues, they report a
plateau or a decline in concentration after a certain time point, suggesting either uptake
saturation or induction of BaP metabolism, or both. Uptake of BaP by Plantago roots
reached a saturation point within 24 hours.Salicornia fragilis grown in oil-
contaminated sediments showed a sharp increase in shoot BaP content between 3 and 4
wks, and a sharp decrease following the 4-wk maximum.^® Both uptake saturation and
uneven rates of metabolism are consistent with observations in animal systems, where
physical limitations on uptake and stepwise metabolism may confound measurement of
linear dosage effects.^’ BaP’s hydrophobicity helps it diffuse passively into membranes
and fatty tissues including roots, and also keeps it bound on soil particle surfaces.
Dispersion by water is confounded by extremely low water solubility, resulting in
environmental persistence and long-term, low level exposures.
In order to dose animal tissues with BaP, DMSO is routinely used with little effect
on the tissues or the organism.®* DMSO was selected as the standard solvent for water-
insoluble test compounds in the Ames assay, due to its excellent solvation properties and
low toxicity.®® Five commonly used organic solvents were shown to decrease BaP
hydroxylase activity in rabbit lung microsomes, but DMSO was the least inhibitory, and
did not have a significant effect at low doses.’® Although DMSO was found to induce
differentiation in cancer cells,"’’^ the difficulty of diluting and dispersing BaP ensures
11 that low concentrations of DMSO continue to be used to dose organisms and cell cultures.” The compromise between the low toxicity of DMSO and its possible interfering effects is to use equal low doses of DMSO in controls and treatments. In plants, the effects of DMSO are mild and concentrations as high as 1% (v/v) have been used to apply lipophilic test compounds.
2.2.3 Selection of plant
Phytoremediation is highly variable by plant family and species. Members of the
Brassicaceae (also called Cruciferae) are well-known for their ability to take up, sequester, and transform metals.’* Crucifers are not noted for their abilities to degrade
PAHs, but no other plant families have been identified as superior degraders of PAHs.
Grasses (Poaceae) often show BaP-remediation potential,’* but this may reflect more
frequent testing based on practical growth factors (diffuse root and rhizome systems,
drought tolerance and easy propagation) rather than inherent degradation abilities. In bench-scale experiments using soil spiked with 100 ppm BaP, celery plants (Apiaceae)
dramatically outperformed wheat, which was not statistically different from control
pots. 77 Members of the Brassicaceae produce distinctive compounds, the glucosinolates,
which are transformed by myrosinase into isothiocyanates. Myrosinase and
glucosinolates are located in separate compartments of the cell, and are mixed together by
cellular damage. Isothiocyanates are generally considered to be produced for defense
against herbivory. The presence of isothiocyanates is a characteristic of the Brassicales
that includes Arabidopsis^^
The therapeutic effects of dietary glucosinolates on mammals have been extensively
researched. Glucosinolates were shown to increase metabolism (activation as well as
12 breakdown) of BaP in cell cultures,’^’*'* and to be chemoprotective against cancer in cell lines*’’*’ and in vivo.^^ Unfortunately, members of the Brassicaceae also contain high
levels of BaP compared with other food plants,*"* and Brassica oleracea acephala var.
Hammer/Grusa has even been used as a bioindicator of BaP pollution.** For these
reasons it seemed likely that Arabidopsis might be able both to tolerate and translocate
BaP. In spite of the tendency to accumulate BaP, foods from the Brassicaceae family
protect humans from carcinogenic activation, therefore the same compounds might
protect the plants themselves from harmful effects of BaP. Arabidopsis was selected
primarily for these reasons, and because commercial microarrays were available with
which to measure gene expression.
A wide range of physical, bioinformatic, and molecular tools (Section 2.3.4) have
been developed and disseminated by the Arabidopsis Genome Initiative (AGI), the
Arabidopsis Biological Resource Consortium (ABRC), and the Arabidopsis Information
Resource (TAIR). These tools, and the advantages inherent in experimenting on plants
rather than cognitional organisms, make Arabidopsis a logical replacement for animal
models in the first phase of research.
2.3 Expression Analysis
2.3.1 Microarrays
As a result of international cooperation in the Arabidopsis Genome Initiative (AGI),
the Arabidopsis genome was published in December, 2000.** Affymetrix produced a
microarray based on the 26,200 annotated genes in the TIGR database on December 15,
2001. The ATHl GeneChip contains more than 22,500 probe sets designed to detect
13 about 24,000 gene sequences. Each gene transcript is represented by a set of 11 different
25-mer oligonucleotide probes, including probes with a deliberate mismatch. For maximum coverage, certain probe sets detect more than one gene. The Greenwood
Molecular Biology Facility (GMBF) at the University of Hawaii at Manoa has acquired the equipment to process and analyze Affymetrix GeneChips. In early 2003, through an arrangement between Monto Kumagai and Affymetrix, the technology was available for the MBBE 680 course, where the initial experiment was designed as a class project.
2.3.2 Quantitative RT-PCR
An accepted method for validating microarray data is the use of quantitative or real time PCR to confirm relative expression levels of a subset of genes. The power of PCR is also its limitation, since exponential amplification can exaggerate miniscule experimental errors. Quantitative PCR has borrowed from analytical chemistry the process of using standards to achieve more reproducible quantitation. As in chemical
analysis, an external standard can provide a fixed reference for evaluating the quantities measured in the sample itself (i.e., compensating for variability of technical factors). An
internal standard relies on the assumption that certain housekeeping or constitutively
expressed genes will have constant expression levels between different samples. Since
constant expression is difficult to ascertain, a better approach is to test multiple reference
genes across all samples in all treatment conditions. Various software programs have
been developed for this, including GeNORM and Normfinder.*^ To correct for saturation
effects of highly expressed housekeeping genes such as actin or ribosomal subunits, it is
best to look for additional reference genes with lower expression levels.
14 The first level of normalizing RNA is to start with equivalent amounts of total
RNA. Because transcription is regulated at multiple levels, and RT-PCR is variable, this is not likely to produce exactly equal amounts of cDNA. Further normalization of nucleic acids from different sample types is accomplished after the reverse transcription of total RNA (the first step of two-step qRT-PCR). During the second step (primer- specific amplification by PCR with SYBR green), inclusion of internal reference genes will allow further normalization of other gene transcript levels.
Quantitative PCR is relatively quick and inexpensive, and is useful for validating small sets of transcripts, and for screening expression of a set of genes in different conditions or timepoints. It is especially useful for comparing relative expression levels, and when used for this purpose is sometimes referred to as semi-quantitative RT-PCR.
Semi-qRT-PCR requires less RNA than Northern blotting, and can provide similar evidence of relative quantities of transcripts in the form of visible bands on a gel.
2.3.3 Expression profiling
The basic understanding of the mechanisms of BaP-induced carcinogenicity has been relatively unchallenged by recent data derived from newer methodology, but the interactions of cells with BaP are still only partially understood. Global expression profiling is a powerful tool for augmenting our understanding and providing a valuable set of putative transcriptional responses to BaP. The transcription responses, especially for genes not previously linked to BaP, must be experimentally verified, and signaling cascades pieced together by comparison and compilation with other experiments, and reverified by further experiments. Analyses that include methylation status of genes and noncoding DNA are also of interest. Proteomic profiling has resulted in new signaling
15 candidates and more accurate estimation of biochemical changes, since it can measure the endpoint molecules at any given time. Phosphoproteomic studies add an extra layer of significance by detailing the phosphorylation status of particular proteins.
Proteins in plant tumors show altered N-glycosylation.^* Glycosylation changes are also found in animal tumors, where distinctive glycosylation patterns in cancerous tissues form the basis for tumor-associated carbohydrate antigen (TACA) vaccine development. 89 Expression profiling may detect differences in the expression of glycosyltransferases and glycosyl hydrolases that result in these altered patterns.
2.3.4 Bioinformatics
The Arabidopsis genome contains 27,235 protein coding genes plus 4749 pseudogenes or transposable elements and 1188 ncRNAs, for a total of 33,282 genes
(TAIR8 release, April 2008). Functional annotation, the primary goal of the Arabidopsis
2010 Project, is still lagging, with approximately half of the genes unknown and/or unassigned in each of the three main GO (Gene Ontology) categories (molecular function, cellular component, biological process)(GO data from May 3, 2007, cited in
April 23, 2008 release statistics).^” In this scenario, sequence-based gene homology remains a major tool for interpreting microarray data, and trying to reconstruct the bigger picture of biological meaning remains slightly beyond our grasp.
Bioinformatics-based comparison of known biochemical pathways in plants and
other organisms can help determine the mechanisms of stimulation or repression by BaP.
Recently, meta-analysis of publicly available microarray data has provided the basis for
assembly of co-expression networks.”' These networks are being incorporated into
16 functional annotation in the ATTED-II database, which also lists over-represented cis elements and transcription factors predicted to regulate the coexpressed genes.
Many useful tools are available through the TAER website, including:
AceView: Compiles expression and functional annotation for a specific gene.^^
AraCyc Metabolic Pathways: Shows association of a gene or locus with known or
hjqiothetical pathways.
ATTED-II: Provides coexpression data compiled from hundreds of microarray
datasets, with over-represented putative promoter motifs and predictions of possible
transcription factors.
eFP Browser: Shows microarray expression data for a given gene in a treatment,
developmental stage, or tissue type.
Genevestigator (https://www.genevestigator.ethz.ch/Server_V3/) Provides analysis
and visualization of compiled microarray datasets.
GO Annotations: Lists Gene Ontology categories for Cellular Components,
Biological Process, or Molecular Function—for single genes or sets of data.
InParanoid Ortholog Groups: Provides orthology summaries for individual genes,
with bootstrap values.
Motif Analysis: Searches for over-represented motifs in a user-defined dataset.
Patmatch: Searches for input amino acid or nucleotide sequences in the proteome,
genome, or a subset such as 1 kb upstream region.
Other web-based bioinformatics tools are available through the central NCBI site:
Entrez Gene: Provides the point of entry for a given gene, with links to other
information about the gene. 17 BLAST: Basic Local Alignment Search Tool, compares sequence homology to other
nucleotide or protein sequences.
BLink: BLAST Link, archives protein-protein BLAST searches to a given protein
HomoIoGene: Lists genes thought to be orthologs of each other.
2.3.5 Gene expression profiling identifies additional genes and pathways
The genetic interactions between BaP and DNA or BaP and P450 enzymes have been extensively investigated. Individual mutations in familiar tumor suppressor genes like P53 were identified long ago, but a comprehensive picture of the entire battery of genes which they regulate has only started to emerge with the sequencing of whole genomes. All published genomes contain many unknown or putative genes; many of these are likely to be signaling molecules or transcriptional regulators. Even when genes have high homology with other transcription factors, their function must be experimentally shown, and the interactions are complicated. In a study using RAGE
(Rapid Analysis of Gene Expression), transcription factor ATF3 was induced and E2A was repressed in early response to BPDE in HME87 normal breast epithelial cells.""' E2A had not previously been reported as a response to PAHs, but the authors hypothesized that it could be responsible for the observed repression of p21-Waf. ATF3 had been implicated in liver dysfunction, so it was not surprising to find that it interacted with
BPDE. Phosphorylation of c-Jun, ATF-2 and SAPK preceded induction of ATF3^'^ which itself down-regulates PEP carboxykinase."* PEP carboxykinase was also down- regulated in TK6 lymphoblastoid cells exposed to BPDE,"* but the authors did not detect
significant changes in ATF3 expression.
18 Advances in large scale profding of transcripts and proteins are producing massive pools of biochemical evidence for the more complex interactions between cells and BaP.
Making sense of all the data will require exhaustive bioinformatic comparisons.
Appendices A and B summarize genes reported as up- or down-regulated in animals in response to BaP or BPDE.
2.4 Related plant research
2.4.1 Plant response to PAH and other xenobiotics
There is little data on genome-wide expression involved in plant response to environmental pollutants. Recent studies have profded plant response to xenobiotic compounds by measuring expression changes in a limited number of conserved genes that are implicated in animal xenobiotic responses. One study measured expression of three genes known or postulated to be involved in stress responses, GSTF2, PRl, and expansinv45, in Arabidopsis exposed to the 3-ring PAH, phenanthrene.®’ The researchers were only able to measure a transcriptional increase in the nonspecific response gene
PRl, and a decrease in ExpA8. In order to demonstrate induction of GSTF2, they employed a line containing 1050 bp of the GSTF2 promoter fused to a IJ-glucuronidase reporter construct.®* In another study, exposure to the explosive RDX induced a 9.7-fold increase in GST in poplar, but other measured genes showed low induction and overlapping error bars, although the authors claim that four additional genes (CYP450,
OPRl, peroxidase, and monodehydroascorbate reductase) passed t-tests for significance at 95% confidence.®®
19 Although glutathione conjugates are exported from the cell in animals, they are targeted to vacuoles in plants. Recently, however, it was shown that gamma- glutamyltransferases (GGTs) degrade the vacuolar GSH-conjugates.’®” Arabidopsis
GGT3 parallels animal GGT as the rate-limiting first step of GSH-conjugate degradation.”” Additional research demonstrated that glutathione-conjugated xenobiotics can undergo long-distance transport and exudation through the root tips of barley.”” This suggests that all three phases of detoxification may be conserved across kingdoms, from oxidation to conjugation, and finally, excretion. The authors also
showed that the transport was unidirectional, ruling out the possibility that exuded
conjugates could be re-imported. They hypothesized that the transporter was
unidirectional. Since MRP-type ABC transporters are well-conserved in eukaryotes and
even some prokaryotes, they could be involved in these processes.
One of the earliest phytoremediation-related studies to use large-scale expression
profiling to investigate plant transcriptional responses was a serial analysis of gene
expression (SAGE) analysis of Arabidopsis root response to trinitrotoluene (TNT).'°*
The authors identified 242 increased and 287 decreased tags putatively representing
genes responding to TNT. Known detoxification genes were significantly up-regulated,
including CYP450s, GSTs, nitrilases, peroxidases, and an ABC transporter. The down-
regulated tags were harder to explain, as little is known about what might lead to down-
regulation responses. Some genes had no known function, and one of the most abundant
tags did not BLAST to any known Arabidopsis sequence, indicating it could represent a
noncoding, miRNA, or siRNA.
20 Baerson et al. 2005 used microarrays to measure Arabidopsis transcriptional responses to the allelochemical benzoxazolin-2(3H)-one (BOA)."*'^ Like the study by
Ekman et al, this study found many homologs to known detoxification genes, and also found altered expression in genes not previously linked to xenobiotic response.
2.4.2 Agrobacterium-mAuceA tumors— a plant cancer model?
The concept of plant cancer has been proposed,'®^ but not widely addressed. There are some similarities between the etiology of plant and animal tumors. At about the same time that early studies of animal tumors posited an important role for viral oncogenes,’®^ a theory emerged that virus-like particles found in tissue cultured plants were responsible for their inability to differentiate."*^ Similar to the findings that BaP interacts with animal hormones in tumor development, (see discussion in Section 2.1.5), BaP concentrations up to 1 ppm increased fern sexual differentiation, and high concentrations of BaP inhibited differentiation."** In marine algae, PAH-contaminated sediment produced tumor-like growths,'**® but the best-studied plant tumors are those induced by
Agrobacterium tumefaciens.
Surprising parallels between Agrobacterium-'inAucQA plant tumors and animal tumors have been identified. Vaseularization is essential for tumor growth in animals, and is a major target of chemotherapy; it plays an important role in rhizobia nodule development and plant tumors as well."** While this research was underway, two studies reported the transcriptome responses of Arabidopsis to Agrobacterium infection. The first study profiled cell suspensions at 48 h after they were inoculated with bacteria.'" The second used whole plants and compared erown gall tissue to non-tumor tissues, and also measured solute profiles."^ Only 7% of the expression changes were shared by the two
21 studies. The expression profile of crown gall tumor tissue resembled a 3-h auxin-induced gene expression profile, reflecting previous measurements of increased auxin and cytokinin levels in plant tumors."^ Ullrich et al. (2000) proposed that overproduction of these plant hormones fills the function of the growth factors that are overproduced during animal angiogenesis. In fact, auxin levels control the development of xylem and phloem,” '' and Ti-encoded genes include auxin and cytokinin synthesis genes, completing the parallel with viral-encoded oncogenes that activate animal growth factor and hormone cascades. Auxin and cytokinin levels are also altered in plant response to
BaP (Forrest 1989), suggesting that comparison of BaP response profiles and crown gall tumor profiles may help clarify the function of differentially expressed genes shared in both processes. Just as the oncogenic viral protein v-Myb was found to have an endogenous cellular homolog (c-Myb),” ^ Agrobacterium-coded genes may have endogenous plant homologs that would be altered when a plant is exposed to a carcinogen.
2.5 Cross-kingdom comparisons
Relevant genes and pathways appear to be well conserved in plants and animals, and sometimes prokaryotes as well. An Arabidopsis annexin gene, ANNATl, conferred resistance to oxidative stress when it was expressed in bacteria. When introduced into
HeLa cells, it protected HeLa cells from TNF-induced apoptosis, and up-regulated native
MnSOD}^^ The ANNATl gene was later shown to have peroxidase activity,” ’ although
the exact functions of annexins in eukaryotes remain unclear. An annexin from Brassica juncea, transfected into tobacco, conferred protection against multiple stresses.”*
22 Heterologous expression can reveal information about gene function that may not be readily observable in its native context. Similarly, by observing gene function across species and kingdoms, we can potentially learn more than we could from one organism.
The power inherent in transcriptome profiling is its ability to reveal previously unknown genes interacting with known responders, to help unravel individual gene function and signaling pathways affected by a stimulus.
23 BaP binds AhR & Arat AhR-Arat BaP—►AhR in —► dimerize— CYP450—►BP DE —► mutations cytoplasm in nucleus bind XRE'
(NFkB) AhR-independent signaling effects; COX-2 not well-defined
B. AhR-dependent effects of BaP
Figure 2.1 (A) Phase I activation of BaP in animals (B) Diagram illustrating AhR-dependent effects of BaP. More than one arrow between labeled steps indicates unknown intervening factors.
24 CHAPTER 3 Hypotheses and Objectives
The mutagenic effects of BaP have been studied in mammalian and prokaryotic systems for over 75 years.” ” In mammals, the binding of BaP to DNA, induction of
CYP450 genes, and repression of tumor suppressor P53 are established mechanisms of mutagenesis. Responses of homologous mammalian genes in plants have not been reported. In addition, an understanding of BaP-induced gene interactions and regulatory networks remains in its infancy. Whole genome microarrays have been used to measure transcriptional changes in cell lines exposed to BaP, helping to identify additional gene expression patterns associated with healthy or cancer phenotypes.’^” Sometimes this sophisticated technology does little more than reinforce previous knowledge, confirming that known tumor suppressors, oncogenes, or molecular markers are appropriately regulated in the tissue sample.’^’ Major progress will occur as meta-analysis of data from many different ‘omics’ experiments begins to help identify gene networks. An organism like Arabidopsis is valuable in that it can not only be subjected to myriad stresses, but can be observed in controlled conditions for optimal study of gene expression within the context of a whole organism.
Comparison of plant responses with those of other organisms should yield interesting differences. It should also reveal similar expression patterns of genes homologous to mammalian genes, that may not be understood in the context of the measured cell line or animal. Emerging understanding of Arabidopsis gene networks can provide insights into gene interactions. These insights will help to clarify why one
25 organism succumbs to lethal effects of a compound, while another thrives and contributes to the degradation of that compound.
3.1 Hypotheses
1. Arabidopsis can take up and degrade moderate to high levels of BaP.
2. BaP elicits transcriptional responses in Arabidopsis similar to responses of
orthologous genes in animals, fungi, and bacteria.
3. The responses oiArabidopsis to BaP have the potential to help clarify the mechanisms
of phytoremediation, phytotolerance, and mammalian cancer.
3.2 Objectives
1. Determine the ability of Arabidopsis to translocate BaP.
2. Determine the ability of Arabidopsis to degrade BaP.
3. Demonstrate that root exposure to BaP can have significant effects on shoot tissue
development (DNA, RNA, and phenotype).
4. Determine the changes in gene expression va. Arabidopsis when grown in BaP.
5. Compare BaP transcriptional responses to other stress responses in Arabidopsis.
6. Compare BaP transcriptional responses m Arabidopsis with other organisms.
26 Chapter 4 Plant Uptake of BaP and Observable Changes
4.1 Introduction
Arabidopsis thaliana is a well-studied model plant with a short generation time, and therefore a good system to study plant interaction with BaP. The interaction of BaP with plants involves a number of interlocking processes. The first step is uptake, followed by translocation, and thirdly cellular interaction, including metabolic and gene expression.
Evidence of uptake could include detection of BaP or its metabolites in plant root, stem or leaf tissue or decreased BaP in the media. Any changes in phenotype would also indicate potential interaction between the plant and BaP. Plant uptake may vary under different growing conditions. I investigated a number of potential BaP exposure routes to enhance uptake, using DMSO, acetone, methylene chloride, and three different media: agar, sand, and hydroponics. The solvents had different effects with different media. In a modified hydroponic system, the BaP was dissolved in acetone and coated onto sand particles, the solvent was evaporated, and then the sand was flooded with aqueous growth solution. In sterile agar culture DMSO was necessary to disperse BaP in the agar.
DMSO was also used in short-term dosage experiments, for fast chemical dispersal to mature plant roots without the risk of transplant shock.
Most of the research on plant response to BaP has focused on the plant’s ability to degrade BaP.'” Studies have demonstrated phytoremediation of BaP in field and in sterile conditions.'” ’” "' Whether remediation is direct (uptake and degradation) or indirect (secretion of factors such as enzymes and/or phenolics into media, with subsequent degradation of the chemical in the rhizosphere) is not known. The specific
27 responses of Arabidopsis thaliana to BaP are unknown. It is crucial to eliminate the possible influence of microorganisms in the media or associated with the plant roots, as this may affect the chemical fate and plant response. In lieu of direct measurement of
BaP or metabolites in the plant tissue (not feasible due to low concentrations), we need to show that any interaction with BaP or its mutagenic metabolites does occur.
Accordingly, an assay for DNA damage (comet assay) was performed.
The alkaline comet assay was used to measure DNA damage in Arabidopsis thaliana plants grown in BaP, as previous studies with BPDE have shown that at least
40% of DNA adducts formed in vitro are at alkali-labile sites.” * Experiments were conducted to explore the effects of media, BaP concentration, solvent, exposure time, and tissue type (root or leaf) on the assay. Plants were grown in sand, liquid MS, or sterile agar media containing 0 to 50 ppm BaP for 24 hours to 6 weeks. Comet assay can quantify DNA damage and allow its characterization as either apoptotic or necrotic. BaP causes DNA damage in mammals, especially at particular regions within tumor suppressor genes, which can lead to cancer.” *’” ’ BaP-DNA interactions are expected to be similar in eukaryotes, but plants sometimes show benefits from BaP exposure,
including growth enhancement.” *’” * The difference may lie in DNA repair, or plants may have higher tolerance for mutations.
4.2 Methods
4.2.1 Growth conditions and phenotypic measurements
Seeds of Arabidopsis thaliana ecotype Columbia were surface-sterilized (agitated in
50% bleach for 10-15 min, rinsed 5x for 5 min each with sterile dH20), then stratified for
28 3 to 5 d at 4° C. Plants were grown in various media: semi-hydroponic in Ottawa sand
(Sigma) or with seedlings growing along agarose-coated microscope slides immersed in liquid media, hydroponic on wire mesh, and in 0.7% solidified agar. Two grades of agar were used: purified agar (Fisher Scientific #A360-500) and Difco Bacto agar (Becton
Dickinson and Company #214050). Media were fortified with 1% sucrose, 0.5x MS
(Murashige-Skoog macro- and micronutrients with Gamborg’s B5 vitamins. Sigma
#M5524). After testing various solvents including acetone and methylene chloride, BaP was dispersed in 0.2% DMSO. Control media eontained equivalent amounts of DMSO.
The optimal DMSO concentration was determined by testing germination in 0.1 to 5%
DMSO; the highest concentration was lethal within 2 days, while concentrations between
0.1 and 0.5% showed no toxicity.
All cultures were grown aseptically. One batch of agar-grown plants was contaminated by a common pink microsymbiont, Methylobacterium. Identity of the bacterium (at the genus level) was confirmed by phenotype and sequencing. Fungal contamination occurred rarely (<1%), usually after sampling of plants, and was easily
observable. After harvest, sterility was verified by streaking media and plant parts on
rich media (TSA, tryptic soy agar), and also on plant methylotrophic endosymbiont-
specific media (Ammonium Mineral Salts, AMS). Sand- and agar-grown plants were
used for phenotypic measurements of root length, leaf length, and length of floral stalk.
Fisher t-tests for significance were conducted in SAS, and graphed in Microsoft Excel.
Germination in agar was assessed by the appearance of root or shoot tissue, without
magnification (see Figure 4.1).
29 4.2.2 Microscopic evidence of BaP effects
Microscopy of more than four week old shoot and root tissue was conducted in the
Biological Electron Microscope Facility at UH (Olympus BX51 fluorescence microscope equipped with UV, PI, and FITC filter sets), and in the laboratory of Dr. David Webb
(brightfield microscopy with osmium tetraoxide staining).
4.2.3 HPLC to detect reduction of BaP in agar
Plants were harvested from 40 mL agar, rinsed with 10 mL H 2 O followed by 15 mL
methylene chloride (CH2 CI2 ). Agar + 25 mL rinsate was stirred 30 min, then centrifuged
at 5000 X g for 10 min at 4 °C. The aqueous fraction was re-extracted by the same method
with two additional volumes of 15 mL CH2 CI2 . The fourth extraction was stirred for 2 h
with 20 mL CH2 CI2 , followed by 15 min centrifugation at 5000 x g.
Extracts were analyzed by HPLC: HP1090 Series II LC, Agilent Eclipse XDB-C8
column (4.6 x 150 mm, 5 pm particle size), water/acetonitrile flow rate 1 mL/min, 25 pL
injection, detection at 254 nm. Gradient started at 50% H2 O : 50% acetonitrile. 3 min
after the injection, the elutant was ramped to 100% acetonitrile and held from 23 to 28
min, then reset to 50% acetonitrile : 50% H2 O for 3 min before the next injection. BaP
eluted at 83% acetonitrile about 15.7 min after injection. BaP (Sigma) was used as the
standard.
4.2.4 Comet assay to measure DNA damage after root exposure to BaP
For testing long-term exposure (4 wks or more), plants were grown in 0.25x MS,
0.5% sucrose, 0.7% agar, with 50 ppm BaP dispersed in 0.2% DMSO, 0.2% DMSO
alone, or with no added solvent. For short-term exposure, seedlings that were started in
agar were transplanted into sterile sand with 0.25x MS -1- 0.5% sucrose. After four weeks,
30 1 mL of liquid 0.25x MS media was exchanged for new media containing 0 to 40 ppm
BaP (w/v) in DMSO (final concentration 0.4%). For consistency, 1 mL of liquid media
in no-solvent controls were replaced with new MS. All plants were grown in a short-day
light regime (100 pE m'^ s’’, 8 h light/16 h dark) at 25 °C, and leaves or roots were
removed immediately prior to processing.
Alkaline comet assay was performed according to Menke et al. (2001).’^^ All
procedures were conducted in low light or using a red safelight. Approximately 100 mg
of leaf tissue (or a smaller amount of root tissue) was placed in a Petri dish on ice, and
sliced with a razor blade to release nuclei into 300 pL of cold PBS + 50 mM
ethylenediamine tetraacetic acid (EDTA), pH=7. An aliquot of 30 pL was mixed with
30-50 pL of 1% low melt agarose (LMA) at 42 °C, and pipetted onto a slide coated with
1% normal melting point agarose (>12 h earlier), and covered with a coverslip on ice.
Coverslips were removed to add 100 pL hot 0.5% LMA, then the coverslips were
replaced, and kept on ice. Slides were placed in ice cold alkali buffer (300 mM NaOH,
5 mM EDTA, pH 13.5), at 4 °C for 5 minutes, followed by 10 minutes of electrophoresis
at 0.7 V/cm in the same buffer at 4 °C. Slides were neutralized in 100 mM Tris-HCl, pH
7.5, for 3 min. The slides were soaked for 10 min in 1% Triton X-100 to remove starch
grains, then rinsed in 70% EtOH and 95% EtOH (2x5 min) and air dried. Gels were
fixed for 10 min in 15% trichloroacetic acid, 5% zinc sulfate heptahydrate, and 5%
glycerol, then washed 2x with dHaO and dried overnight at RT. Silver staining followed
a protocol validated for consistency across seven different labs.'^” Slides were rehydrated
for 5 min in dH20, followed by 35 min in the dark with agitation in a Coplin jar
containing 66 mL of 5% sodium carbonate mixed with 34 mL of 0.1% ammonium
31 nitrate, 0.25% tungstosilicic acid and 0.15% formaldehyde (v/v). Slides were rinsed 3x in dH20, then placed for 5 min in 1% acetic acid to stop the reaction. After air-drying, the slides are stable indefinitely. One hundred nuclei per sample were scored visually under light microscopy at 400x by placing them into one of the three categories depicted
in Figure 4.6. The categories were: comets, intact nuclei, and ‘hedgehogs’. Hedgehogs
are nuclei with diffuse tails separated from small heads, and indicate aUcali-labile DNA
diffusion during the alkaline lysis incubation step. All slides were randomly coded to
ensure unbiased scoring.
4,3 Results
4.3.1 Phenotypic changes in Arabidopsis following exposure to BaP
Seeds sown directly on agar containing BaP germinated about two days earlier than
on control agar with 0.2% DMSO (Figure 4.1). Plants grown in 50 ppm BaP in Fisher
purified agar had healthier and more extensive root systems after months of culture,
thinner stems and shorter leaves, and lived more than twice as long as DMSO-only
controls. However, when plants were grown in tissue culture grade agar (Difco Bacto
agar), all plants lived about the same length of time. Hydroponic plants lived longer
when grown in BaP, but sand-grown plants did not show such a distinct difference in
lifespan.
Sand-grown Arabidopsis had longer leaves and roots when grown in 40 ppm BaP.
Variances were pooled after the test for homogeneity of variances showed we were not
able to reject the hypothesis for equality of variance. The range of leaf lengths was 0.4 to
1.7 cm. The mean leaf length in BaP plants was 1.13 cm (5D=0.29), and 0.88 cm
32 (5D=0.26) in control plants. There was a significant increase in leaf length with BaP exposure, t (40) = -2.97, p = 0.005. Root lengths ranged from 0.4 to 5.0 cm. Mean root length in BaP plants was 2.65 cm (5D=1.13), and 2.03 cm (SD=1.06) in controls. There was a slight increase in root length with BaP exposure, t (40) = -1.86, p = 0.07. BaP- treated plants displayed greater variation in floral shoot length and timing of floral induction, but the averaged variation was not significantly different from controls (data not shown).
Exposure to BaP in sterile agar, sand, or hydroponic solution produced measurable phenotypic differences, indicating that Arabidopsis interacted with BaP, independent of microbial influence.
Table 4.1 T-test for phenotypic differences between plants grown in sand with 40 ppm BaP or controls with 0.4% DMSO. There was a significant increase in leaf length with BaP exposure, t (40) = -2.97, p = 0.005. BaP exposure slightly increased root length, t (40) = -1.86, p = 0.07.
Mean Length (cm) SD P Control Leaf 0.88 0.26 0.005 BAP Leaf 1.13 0.29 Control Root 2.03 1.13 0.07 BAP Root 2.65 1.21
4.3.2 Microscopic evidence of BaP effects
Differences between control and treated shoot tissue were difficult to determine
with microscopy. Root tissues, however, showed some clear differences. UV-
fluorescent bodies consistent with BaP were clearly visible in and around roots exposed
to BaP, but not in controls (Figure 4.2). Control roots showed bodies that fluoresced red
33 under FITC filters. These were not generally seen in BaP-exposed roots. Osmium tetraoxide (lipophilic) stain revealed greater numbers of dark yellow-colored bodies in the BaP-grown roots (Figure 4.3), which could correlate either with BaP metabolites or with the apparently phenolic compounds that turned spiked agar yellow-orange.
4.3.3 HPLC to detect reduction of BaP
HPLC results show a decrease in BaP concentration in planted vs. unplanted agar.
Control agar stored in darkness showed there was also a decrease in unplanted agar in the
light (Figure 4.4).
4.3.4 DNA Damage Detected by the Comet Assay
Under alkaline assay conditions, all samples had a high level of ‘hedgehogs’.
Figure 4.7 shows that leaves of Arabidopsis grown for 4 wks in agar containing 50 ppm
BaP had a significant increase in DNA damage, compared to both DMSO and no-solvent
controls. In plants exposed to 8 ppm BaP for 24 h, root nuclei showed more than a nine
fold increase in long, narrow comets, compared with all other samples (Figure 4.8). No
significant difference was found in leaves exposed to different doses of BaP (0.8, 8, and
40 ppm, in sand) for 24 h. DNA from leaves grown in 0.4% DMSO produced fewer
hedgehogs than all other treatments, at both exposure times.
34 Figure 4.1 Stratified seeds germinated faster on Difco Bacto agar with 50 ppm BaP in
0.2% DMSO than on agar with DMSO alone.
35 Figure 4.2 Comparison of fluorescent bodies in root tissue. Fluorescent bodies are visible within and around BaP-treated root (left) but not control root (right).
36 A. DMSO control root B. BaP-exposed root
Figure 4.3 Osmium tetraoxide-stained roots. BaP-grown root contains many dark yellow spherical bodies. These may be oil bodies with or without BaP, or vesicles containing lipophilic compounds. Most bodies appear to be in the phloem, suggesting they could be membrane-bound transport vesicles for the yellow-orange compounds observed in BaP spiked agar after 3-4 weeks of plant growth.
37 Late harvest
No plants, light
No plants, dark
1 1 1 1------20 40 60 80 100 120 % recovery, compared to dark control
Figure 4.4 HPLC results showed a decrease in BaP concentration in planted agar vs. unplanted control agar. Agar was extracted 4x in methanol. Data were plotted based on comparison with a standard curve, and expressed as % recovery, with the dark control extract = 100%.
38 1
J
Figure 4.5 Darkening of the media and roots in the presence of BaP.
Color change may be due to photoprotective phenolic compounds such as carotenoids, which might be secreted into the translucent media, analogous to the yellow color of pollen which helps to protect it from UV damage. Phenolic compounds are known to be secreted by plant roots.
39 comets intact nuclei ‘hedgehogs’ ►
Figure 4.6 Types of nuclei observed in alkaline comet assay. Arrow indicates direction
of DNA migration.
40 DNA Damage Measured by Comet Assay 100 n 90 - 80 - 70 - % 60 □ intact nuclei □ alkali-labile DNA 1^0 40 - ■ DNA damage 30 -
20 -
10 - 0 I. control DMSO BaP
Figure 4.7 Plants grown in agar with 50 ppm BaP showed two significant differences: fewer hedgehog nuclei (from alkali-labile DNA) than control and DMSO-grown plants, and more comets (reflecting DNA damage and/or cross-linking). The comet assay showed no significant differences between the two controls, except that nuclei from plants grown in 0.2% DMSO contained less alkali-labile DNA than nuclei from non solvent controls. Error bars represent standard deviation from the mean, n=3.
41 24-h BaP Exposure in Sand-Grown Plants
100 - 90 - 80 - O) o 70 - □ intact DNA c 60 - □ hedgehogs B 50 - o 40 - ■ comets o 30 - 20 - 10 - 0 - X Control 0.4% 0.8 ppm 8 ppm 40 ppm 8 ppm Leaf DMSO BaP BaP BaP BaP Leaf Leaf Leaf Leaf Root
Figure 4.8 Comet assay of sand-grown Arabidopsis shows individual differences in
DNA damage within 24 h of exposure to BaP. Each bar represents 100 nuclei. The highest levels of damage are in root tissues, which were directly exposed. Note that 0.4%
DMSO had a greater protective effect than 0.2% DMSO in Figure 4.7.
42 4.4 Discussion
Measurement of changes in BaP concentration in media, coupled with biological confirmation that no microbes were present indicate that plants either took up or absorbed
BaP, or produced root exudates which helped degrade it.
A decrease in BaP concentration occurred in the growth media. The average decrease was 15%, or 300 pg BaP in 40 mL of media, over a four-week growth period.
When plants were harvested earlier, the concentration of BaP remaining in the agar was lower than from agar where plants were allowed to remain longer. This leaves open the possibility that plants may initially take up the compound, but may also have the ability to secrete it or its metabolites through the root system. Arabidopsis plants secrete many different ring-containing compounds in response to various stresses or elicitors,'^’ such as phenolic compounds in response to PAH exposure.It is possible that these compounds are metabolized PAH products. We were unable to detect BaP or proximate metabolites in plant tissue due to low concentrations, with ppb levels reported in other plants,” ^ and the overall small plant biomass available from Arabidopsis. However, a decrease in BaP concentration was measured in planted versus unplanted agar. This is consistent with previous research”'* which shows that planted soils have lower concentrations of BaP than unplanted control soil.
Some of the interactions with BaP may have reached saturation. For example,
uptake depends at least in part on passive adsorption to the root, and it is possible that the
surface was saturated before the fourth week. Brady et al. (2003) demonstrated that
radiolabeled BaP aggregated on the surface of Plantago roots within 24 h, but no further
uptake was evident over seven days, and no translocation to shoots was visible in
43 autoradiographs.’** In Arabidopsis, it was possible to see a dark yellow color consistent with BaP quinone metabolites on the surface of the roots without using radiolabels or fluorescence (Figure 4.5). In addition, by 4 weeks planted Fisher brand agar showed visible darkening, ranging from dark yellow to orange. Quinone products from BaP would have a brownish color, but methanol extracts of the agar did not co-migrate with
BaP or quinone standards when examined by thin-layer chromatography (TLC). The color change in the agar is consistent with phenolic root exudates.
Microscopic observations were consistent with BaP-related changes or metabolism, but without further confirmation with specific probes, it is unclear what occurred in the plants. I tried using an anti-BaP antibody (mAb 4D5)’** with fluorescent secondary antibody to detect BaP uptake in the tissue, but the low shoot concentration and high background fluorescence did not provide definitive answers. With both confocal and
fluorescence microscopy the bandpass filters were too wide to eliminate the background
fluorescence. Plant tissues have lignin, which fluoresces in the UV range, chlorophyll,
which fluoresces in the FITC and rhodamine ranges, and many phenolic compounds
overlap with the fluorescence of BaP. At higher magnification, using TEM or SEM and
gold-labelling, the antibody could have been very useful.
Studies with human fibroblast cells or murine macrophages successfully used
benzo[a]pyrene fluorescence to monitor uptake and localization.’*’ These cell types have
lower background fluorescence than whole plant tissues, and the authors used multiple
fluorophores and filter sets to determine colocalization in subcellular compartments. The
microscope also had narrower bandwidths for excitation, and optimal emission filters. A
more sophisticated study of PAH uptake by fungal hyphae also benefitted fi-om the low
44 1 Tft native fluorescence of the tissue. The authors were able to show the storage of BaP in lipid vesicles, using a combination of specific excitation and emission filter sets and broad-range images analyzed with special 3D imaging software (Simple PCI software
from C-Imaging). With more specific filter sets and additional optimization, it might be
possible to visualize the BaP uptake in plants, especially if the uptake levels could be
artificially augmented by intense short-term application of concentrated chemical.
The long, narrow comets characteristic of root DNA exposed to BaP (90% of the
nuclei, versus less than 10% in leaf tissue) may reflect inhibition of denaturing via cross-
linking of DNA."® Comets are produced by two steps in the assay. First, the DNA is
relaxed during the stationary unwinding step in alkali buffer. This is analogous to the
alkaline elution assay, which produces nuclei with halos of relaxed DNA."** Second,
DNA that may have been less labile in alkaline treatment alone, is subjected to an
electrophoretic field, which moves smaller and/or single-stranded DNA more efficiently.
The dramatic decrease in hedgehog nuclei in DMSO-grown plants may be due to
the antioxidant effect of DMSO."' While DMSO plants showed a similar phenotype to
the no-solvent controls, trace levels of DMSO in the tissue may have protected the
nuclear DNA before and during the assay. Generally, hedgehog comets are taken as
evidence of more extensive DNA damage,'"*^ and are often considered markers of
apoptosis.'*** However, there is considerable controversy about scoring them this way,
and some researchers choose not to score them at all.'" Many researchers regard them
as a logical progression from less damaged comets, as the shorter-tailed comets also have
a fairly wide footprint. In this study, the high background levels of hedgehogs was not
surprising due to the harsh eonditions used (highest pH, and highest levels of EDTA), and
45 also were not particularly informative. Because the long, narrow comets were so distinctly different from hedgehogs, it was easy to recognize the difference in the various
BaP exposures. This comet phenotype may reflect resistance to alkaline lysis and dispersal during incubation due to cross-linking of DNA exposed to BaP. Other mutagens have similar effects,'"*^ and this method may prove useful for scoring biologically relevant differences not captured by a simple tail length measurement.
Traditional scoring methods include tail length, percentage of DNA in the tail, and Olive tail moment, which accounts for both parameters.’"*®
We demonstrated that BaP causes DNA damage detectable by the comet assay. The highest levels of damage occurred in roots directly exposed to the chemical. The data
indicated that Arabidopsis may be able to translocate BaP and/or its mutagenic products
after long exposures. In addition, a novel scoring method was proposed, that solved a
problem frequently encountered with the alkaline comet assay, the high background
migration of control DNA.’"*’ Higher numbers of “hedgehogs” are sometimes reported in
control nuclei than in those treated with known mutagens.’"**’’"’^ Early plant comet assay
techniques (modeled on animal assays) used up to 100 mM EDTA for lysis and/or
slicing,’®”’’®’ but these high concentrations of EDTA, particularly in the high pH buffer,
may cause oxidation of exposed plant nuclei. Recent protocols have called for decreasing
the amount of EDTA, and addition of 5-10% DMSO in the isolation buffer to reduce
background oxidative damage.’®^ However, this method, which measured DNA damage
in plants grown in BaP + DMSO, DMSO alone, or media with no added solvent, was
sensitive enough to show differences between DMSO and no solvent controls. The
46 decrease in hedgehog nuclei in DMSO-grown tissue might have been obscured by the use of a protocol that included DMSO in the preparation buffer.
47 CHAPTER 5 BaP Alters Gene Expression of Plants Grown in BaP for 4 Weeks
5.1 Introduction
Affymetrix ATHl GeneChips were used to compare gene expression profiles in shoots of Arabidopsis plants grown with or without BaP. Differences in gene expression profiles after 4 weeks of exposure were expected to suggest mechanisms of BaP degradation and tolerance in plants relevant to phytoremediation and cross-species carcinogen response. Quantitative PCR was used to measure expression of selected transcripts from plants grown in non-sterile soil to confirm microarray results and ensure that response is related to BaP rather than DMSO or other growth conditions.
At the time of this study, global expression profiling of plant response to BaP had not been reported. Analysis of sterile shoot tissue targets plant genes involved in later interactions with BaP. Root analysis would more likely pick up genes involved in early transport, rhizosecretion of enzymes and phenolics. However, if initial processes occurring in roots (binding, oxidation, conjugation, transport) predominate, differences in shoot gene expression may be either incidental or represent secondary transport and sequestration processes plus a generalized defense response. The experiments were expected to yield promising candidate genes for further investigation of BaP-plant interactions. They also hold the potential to confirm or clarify patterns observed in animal responses to BaP.
The purpose of measuring the gene expression of sterile-grown plants was to control the experimental parameters and limit the results to plant-BaP interactions. In order to assess the applicability of the research results to phytoremediation, the
48 experiments were repeated using plants grown in non-sterile soil conditions. It was possible that many of the expression patterns would not be validated in the soil experiments, as even small fluctuations in light and temperature within a single growth room or even among neighboring plants in a single cohort could result in measurable changes in gene expression. However, it was also considered that this could be a means of reducing the number of gene changes in the data set. Decreasing the number of genes included in the analysis might help to ensure that the most significant or essential genes were not overlooked due to the sheer volume of data. Because ‘-omics’ technologies are relatively recent options, scientists have tended to rely on traditional experimental approaches that seek to minimize variability in measurements. Although generally a good scientific practice, strict control of conditions may overlook the inherent value of variation. The power of the microarray is its ability to collect vast amounts of data; this can also be a drawback, since the human mind cannot fathom an entire data set.
Therefore, altering experimental conditions can potentially be more informative than standard replication, since experimental variation can be used to whittle down the data pool, leaving only the most robust effects.
It was also important to ensure that any extrapolation to phytoremediation conditions was valid. Previous experiments with semi-hydroponic growth in sterile sand showed that plants were much healthier in a semi-solid substrate than in hydroponic culture (data not shown). Generally, BaP-exposed plants were healthier than controls when both were grown in sand (but none of the plants thrived in the semi-hydroponic
conditions). Leaf size and germination rates were higher in the presence of BaP (data not
shown). The growth differences between bleach-sterilized seeds grown in sterile agar
49 and plants grown in soil with circulating air illustrate the huge effects of factors like hypoxia, nutrient limitation, and symbiotic bacteria. Soil-grown Arabidopsis may grow to over 18 inches in height, while plants cultivated in agar in enclosed culture jars rarely reach half that height.
A major advantage afforded soil-grown plants was possible colonization by
symbiotic or commensal Methylobacterium species, some of which have been shown to
produce cytokinins that stimulate plant growth. The presence of symbiotic
methylobacteria can greatly increase plant size and vigor, as seen in Figure 5.1.
Methylobacteria may be involved in PAH degradation,"® and have even been observed
growing in direct contact with crystals of phenanthrene (a 3-ring PAH)."® Therefore,
soil phytoremediation studies are likely to result in more effective degradation of PAHs,
due to improved plant vigor and additional microbial degradation. Huang et al. (2004)
compared degradation of mixed PAHs in creosote (a coal tar distillate), with bacteria
alone, with plants, and with plants supplemented with plant growth-promoting rhizobia
(PGPR)."’ They found that plants degraded larger PAHs, resulting in the degradation of
20% of the BaP. The addition of PGPR increased the degradation to over 50%, but the
soil bacteria alone were not able to remediate BaP significantly. The enhanced
degradation was attributed to increased plant biomass due to stress reduction by PGPR,
which impact ethylene production by metabolizing the precursor (ACC)."’ A tolerance
for higher concentrations of creosote was also observed, if the plants were inoculated
with PGPR before exposure to creosote."*
50 5.2 Methods
5.2.1 Plant growth conditions in agar
For microarray analysis, plants were grown in 1% agar (Fisher Laboratory Agar), fortified with Ix Murashige-Skoog basal salts, (w/v), and either 50 ppm BaP in DMSO
(0.2% final concentration, v/v) or 0.2% DMSO only. Agar was autoclaved, then spiked with BaP or solvent only. All jars were reheated in a microwave to aid in dispersal.
Seeds oiArabidopsis thaliana ecotype Columbia were surface-sterilized (agitated in 50% bleach for 10-15 min, rinsed 5x for 5 min each with sterile dH20), then plated on 1% agar with Ix MS and 14% sucrose. Seeds were stratified for 1 d at 4° C in the dark.
Seedlings were transplanted to sterile tissue culture jars 1 wk after germination, and grown with a 9 h light/15 h dark cycle under cool white fluorescent lights, at -100 pE m'^ s” . Tissue was harvested at 4 wks, taking mature but non-flowering rosettes at growth stage 3.9 according to the classification system developed by Boyes et a/.” ”
5.2.2 Affymetrix ATHl GeneChip analysis of agar-grown plants
Roots were excised and rosettes were flash frozen in liquid nitrogen and stored at -
80° C. For RNA extraction, approximately 100 mg of frozen shoot tissue were ground in an RNase-free 1.5 mL microtube in liquid nitrogen, using a blue plastic pestle (Research
Products International, Mt. Prospect, Illinois). Total RNA was extracted from 3 control and 3 treated samples using Qiagen RNeasy Plant Mini Kit. Because the first buffer in this kit contains guanidine isothiocyanate (GITC), which might react with cytochrome
P450 enzymes induced by benzo[a]pyrene,’®” the alternative buffer RLC (contains
guanidine hydrochloride) was used. After this buffer was added to the sample, it was protected by the included RNase inhibitors and could be further homogenized. One
51 hundred milligrams of fresh shoot tissue yielded between 20 and 50 gg of total RNA, quantified by comparative absorbance at 260 nm and 280 nm and analyzed on Agilent
2100 Bioanalyzer. Each RNA sample represented a distinct pool of non-flowering rosette tissue from at least six individual plants.
After reverse transcription, 15 to 20 gg of cDNA from each sample were hybridized to Affymetrix ATHl GeneChips. Data were analyzed using Affymetrix software
Microarray Suite (MAS 5.0), with global scaling normalization by the GCOS algorithm, and Welch’s t-test with significance set at 0.05. Pairwise comparison of normalized gene expression levels (treated/control) was performed by Data Mining Tool and
MicroDB, and genes called ‘increased’ or ‘decreased’ in at least 8 of 9 comparisons were compiled into the short list of expression changes. The full data set was further analyzed using Microsoft Excel (2000 and XP), to remove genes with too many ‘absent’ calls
(greater than 2 of 6 called absent) or illogical conclusions (i.e., called ‘increased’ but absent in 2 of 3 treated samples). To generate the larger list of gene expression changes, a fold-change cutoff of 1.5x was used.
5.2.3 qRT-PCR of plants grown in soil
Gene Selection
Genes were selected based on microarray results. Although many of the more interesting genes were expressed at low levels, transcripts with higher levels of detection were selected, to make it more likely that differences could be measured by qRT-PCR, and could be detected in samples taken from plants grown in different conditions (soil and constant light).
52 Plant Culture and Experimental Design
To ensure the previous experiment was reproducible and applicable to more realistic situations, Arabidopsis were grown in soil, without aseptic conditions. To eliminate background microbial differences. Supersoil™ potting soil (Scott’s) was autoclaved in 400 mL glass beakers, 1 h per day for 4 days. Soil was wetted with BaP dispersed in HPLC-grade acetone, or an equivalent amount of acetone for the control pots, and the solvent was evaporated in a fume hood over 4 days. The final concentration of BaP was approximately 50 mg/kg (dry weight). Soil was re-wetted with 0.25x
Murashige-Skoog basal salts (Sigma-Aldrich Corporation), and Arabidopsis thaliana
Col-0 seeds were sown on the surface. Beakers were placed in a fiime hood with constant light of about 800 lux, in a room kept at 23 to 25° C. Plants were harvested at growth stage 3.9, about 26 d after germination, and flash frozen in liquid nitrogen.
Frozen rosettes were kept at -80° C until used for RNA extraction.
RNA Purification
Soil-grown plants were ground in baked ceramic mortar and pestles, since more tissue was available. RNA was extracted from approximately 100 mg of ground shoot tissue with a Qiagen RNeasy Plant kit, using the RLC buffer. Additional homogenization was done after adding the RLC buffer containing RNase inhibitors, and before pipetting the sample onto the initial shredder column. All washing steps were performed, and columns were eentrifiiged 1 additional min in new tubes, to remove residual ethanol.
RNA was eluted in 2 volumes of 30 pL RNase-free water. After purification, total RNA
was DNase-digested off-eolumn for 30 min at 32° C using DNase I and Protector RNase
inhibitors (Roche). Samples were repurified by adding 350 pL buffer RLC and 250 pL
53 95% EtOH, and pipetting onto a Qiagen RNeasy purification column. The column was washed with 500 pL Buffer RWl by centrifuging at 8000 x g for 15 s, followed by a
second volume for 2 min. RNA was again eluted in 2 x 30 pL H 2 O. Total RNA was quantified with a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington,
Delaware).
Primer Design & PCR
Primers were designed using various web-based tools. Occasionally, primer sequences from the Roche Universal Prohe Library (https://www.roche-applied- science.com/sis/rtpcr/upl/adc.jsp) were used without modification. These primers usually amplify a region that spans an intron, resulting in a larger amplicon for gDNA.
To check this, each primer and amplicon was BLASTed to check for homology to DNA, and amplicons were aligned with the unspliced gene sequence to locate introns.
Unspliced sequences are available in the MIPS database, hyperlinked from TAIR locus records, or available directly at http://mips.gsf.de/proj/plant/jsf/athal/searchjsp/index.jsp.
When possible, primer pairs were designed so that at least one primer spanned an intron:
1. The unspliced gene sequence was downloaded from the MIPS database.
2. An intron-spanning sequence was selected and pasted into a primer design
program: Genscript Primer Design Tool (http://www.genscript.com/cgi-
bin/tools/primer_genscript.cgi) or Invitrogen OligoPerfect Designer
(http://tools.invitrogen.com/content.cfm?pageid=9716).
3. Input options and sequence were manipulated until one primer spanned an intron.
54 4. Each primer was BLASTed against nucleotides from A. thaliana in the NCBI
‘nr/nt’ database, to check for non-target amplification in both DNA and RNA.
The default option was modified to search for ‘somewhat similar sequences’
(BLASTn) {http://ncbi.nih.gov/BLAST/).
5. Secondary structures due to self-annealing or heterodimers were checked using
IDT OligoAnalyzer software at;
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/.
Primers were synthesized by Integrated DNA Technologies (Coralville, lA) through a contract administered by the Biotech Core Facility (GMBF) at UH. Primer specificity was confirmed using the RT-PCR product in a PCR reaction with Eppendorf Master Mix or Roche Expand High-Fidelity PCR reagents and gene-specific primers. Products were run on 3.5% low-melting agarose gels in Ix TAE buffer at 90-100 V, to check for bands corresponding to genomic DNA amplicons, RNA amplicons, or primer dimers. Roche primer sets usually span an intron, so that genomic DNA will either not be amplified, or any gDNA product will be visible on a gel as a larger band. Sometimes nonspecific products will be visible in the melt curve analysis. Whenever possible, primer sets were designed so that one primer spanned an intron, to ensure that genomic DNA would not amplify. To check the quality of individual RNA extracts, samples were reverse-
transcribed, then primers that produced both an mRNA amplicon and a genomic DNA
amplicon were used to amplify cDNA products. The PCR products were then run on a
gel to assess whether RNA needed further DNase treatment.
55 qRT-PCR
Gene expression levels were measured by two-step quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). In the first step, I to 2.5 pg total
RNA was reverse-transcribed using a Bio-Rad iScript cDNA synthesis kit. At the same time, no-RT control samples were made using the iScript cDNA synthesis kit, except an
equal volume of water was substituted for the reverse transcriptase. Quantitative RT-
PCR was performed on 20 ng aliquots of cDNA, or equivalent no-RT control samples, in
20 pL reactions with iQ SYBR green Supermix (Bio-Rad, Hercules, CA) on a Bio-Rad
IQ5 Cycler. Primer concentrations ranged from 125 nM to 500 nM, after optimization.
Melting temperatures were 59° or 60° C. The IQ5 Cycler was programmed to run 3 min
@95° C for enzyme activation, followed by 40 cycles of 15 s @95° C, 15 s @59° or 60°
C, and 15s @72° C. A melt curve analysis was also performed, with 0.6 s increments of
0.5° C, from 59° C to 90° C. Expression levels were calculated from background-
subtracted IQ5 data, using Real-time PCR Miner algorithm,’^’ and normalized to the
mean of actin 2 (At3gl8780) and tubulin 8 (At5g23860). Real-time PCR Miner results
are comparable to analysis by delta-delta Ct, DART-PCR and LinRegPCR algorithms for
efficiency correction.’^’ After the PCR, reactions were run on a 3.5% agarose gel in Ix
TAE (90-100 V) to check for non-specific amplification products.
5.2.4 Bioinformatics and data mining
Bioinformatics Tools
Many useful applications are publicly available through the internet, including
many provided through the TAIR website.’®’ The TAIR Patmatch and Motif Analysis
tools were used to search for over-represented peptide motifs and promoter motifs. The
56 eFP Browser and Genevestigator tools (linked through TAIR) were used to look up microarray results for single-gene expression levels in a developmental stage, treatment, or tissue type.
The Gene Ontology (GO) Annotation tool made it possible to visualize large scale expression changes characterized by the three categories of cellular component (Figure
5.3), biological process (Figure 5.4), or molecular function (Figure 5.5). In order to ensure that there were sufficient numbers of genes to make relevant assessments of differential expression of genes corresponding to each of the GO categories, analyses were made using two different large data sets. The GO analyses depicted in Figures 5.3-
5.5 used a data set with expression ratios of 1.4-fold or greater, consisting of 224 genes that were 1.4-fold up-regulated in BaP-exposed plants, and 281 genes that were down- regulated. The down-regulated genes were input as the Control genes, since they were higher in control plants with respect to BaP. In both cases, GO annotation was only available for 82 to 92% of the genes in the Biological Process, Molecular Function, and
Cellular Component categories, so percentage representation in control or BaP- responsive genes was calculated from slightly smaller, but comparable numbers. An analysis using only the genes called ‘increased’ or ‘decreased’ in eight of nine comparisons produced similar results, but was less robust due to low numbers of genes in the subcategories. An analysis using 959 BaP/up-regulated and 635 Control/down- regulated genes agreed substantially with the GO analyses from the two smaller data sets,
confirming results shown in Figures 5.3-5.5.
AraCyc metabolic pathway tools allowed investigation of function within the
context of a metabolic pathway. ATTED-II coexpression data was used to verify patterns
57 of coexpression in the 24-h response data.’*"* hiParanoid Ortholog Groups provided bootstrap values for orthologs that were also checked by BLINK (protein BLAST searches hyperlinked from NCBI protein records) and HomoloGene (NCBI-hosted gene homolog database). AceView was used to cross-check functional annotation, homology, and expression data for a specific gene.
5.3 Results
5.3.1 Phenotypic observations
A slight but insignificant increase in leaf size was observed (data not shown) in plants grown in soil with BaP versus control plants. Arabidopsis plants were generally much healthier in soil than in sterile agar. As seen with the beneficial effect of methylobacteria contamination of one batch of agar-grown plants (Figure 5.1), soil microbes probably contributed to the healthier phenotypes. Root aeration was undoubtedly improved in soil.
5.3.2 Microarray expression results
For different purposes, the data were variously expanded and contracted by adjusting the minimum cutoff parameters. The minimal data set included 75 probe sets that were called increased, and 51 probe sets that were called decreased in all replicate pairs. The probes represent 80 and 57 loci, respectively, and are shown in Table 5.2.
Microarray analysis that set a cutoff at fold changes of 1.5x identified probe sets representing 179 up-regulated and 161 down-regulated loci; these are listed in Appendix
C. When the set was expanded to include gene changes as low as 1.4-fold, there were
224 up-regulated genes, including 11 ‘extra’ genes as two genes were targeted by the
58 same probe set in 11 instances, and 281 down-regulated probe sets, including 15 ‘extra’ genes. This analysis was the only one in which the genes identified as down-regulated outnumbered the up-regulated genes, possibly indicating that there were more false positives in this set.
5.3.3 Variation among the same class of genes
A total of three nudix hydrolases (Nudt) were down-regulated by BaP exposure, and the transcript that had intermediate confidence levels was confirmed by qRT-PCR of soil- grown plants. Nudt21 and Nudt? were decreased in microarray analysis of BaP-exposed
Arabidopsis, but with variable responses in individual (pooled) samples. The Nudt? expression ratios for three paired microarray samples were -1.2, -1.4, and -2.1 fold.
Nudt21 ratios were more significant, at -1.4, -1.9, and -2.6, but showed the same pattern across the three paired samples, indicating that these two genes may be coregulated.
NudtS was called decreased in only two of three paired samples (the middle pair showed a slight increase in BaP, compared to the corresponding control).
In the qRT-PCR of the soil-grown plants, Nudt? expression was also variable in different samples. The average normalized expression of four BaP-exposed samples was the same as that for the four controls: 1.9. If individual samples are compared to the average, expression of the controls was 2.2, 1.3, 1.3, and 1.0 (average) times higher than the average. For BaP-treated plants, expression was -1.9, -17.3, 2.3, and 1.1 times the average, indicating that BaP had a distinct repression effect on two of the samples.
5.3.4 qRT-PCR results
The expression patterns of 9 out of 10 genes selected from microarray results were confirmed by qRT-PCR of soil-grown plants (Figure 5.7). The magnitudes of the
59 changes were not precisely duplicated, but only one gene, GDE, showed both an opposite trend and less than significant fold change (Figure 5.6). A second gene, the multidrug- resistant protein homolog MRP2, was not as clearly down-regulated in the soil-grown plants as it had been in the microarray experiment.
The gene that was not validated was GDE (At5g41080), which has 27% identity and 46% similarity (E=4e-18) to human glycerophosphodiester phosphodiesterase GDE5.
Mammalian genes that contain the glycerophosphodiester phosphodiesterase domain are up-regulated during differentiation."^ Down-regulation of this gene measured by microarray in agar-grown plants was not confirmed by qRT-PCR, which showed moderate but not significant up-regulation when plants were grown in soil (Figure 5.7).
Down-regulation in agar could reflect suppression of differentiation, which is consistent with a carcinogen response that produced the static green condition observed in long- lasting shoots that were grown in sterile agar. The slight up-regulation measured in soil- grown plants is consistent with their healthier appearance and normal lifespan. Although it is possible that the microarray result was a false positive, it appears that this gene is differently regulated in BaP-treated plants as compared with controls, and the conflicting results reflect altered experimental conditions.
The changed conditions included eight different factors, mostly related to the strictly controlled, sterile growth conditions in the first experiment, which had to be altered in order to provide a more realistic environment. The use of non-sterile soil in open containers made it necessary to place the plants in a chemical fume hood. It was not possible to fit the hood with lighting that could be controlled by a timer, which meant that
plants received constant light instead of short-day conditions. The major benefit of
6 0 switching to soil was that the solvent could be eliminated by spiking the soil with BaP in acetone, which was evaporated completely before introducing plants. A full comparison of the different experimental conditions is given in Table 5.1.
61 Table 5.1 Differences in the experimental conditions in the two 4-week growth experiments
Microarray qRT-PCR
Agar Soil
DMSO, 0.2% Acetone, evaporated
Sterile Non-sterile
High humidity Low humidity (air conditioning)
No air flow (hypoxia) Strong air flow (fume hood)
Short-day Constant light
Late day harvest Earlier harvest
Transplanted to BaP Germinated in BaP
62 Table 5.2 Genes called all Increased or all Decreased in GeneChip. Loci in bold were checked by qRT-PCR. Probe sets hybridizing to more than one gene are listed once.
Array Locus FC Element Identifier Annotation AT5G38930 germin-like protein, putative 6.9 249477_s_at AT5G38940 manganese ion/metai ion binding / nutrient reservoir AT3G59930 Encodes a defensin-like (DEFL) family protein. 6.5 251438_s_at AT5G33355 Encodes a defensin-like (DEFL) family protein 5.8 254889_at AT4G11650 ATOSM34 (OSMOTIN 34) 5.5 260386_at AT1G74010 strictosidine synthase family protein 4.9 259478_at ATI G18980 germin-like protein, putative 4 260101_at AT1G73260 trypsin and protease inhibitor / Kunitz family protein 3.8 254543_at AT4G19810 glycosyl hydrolase family 18 protein 3.8 259553_x_at AT1G21310 ATEXT3 (EXTENSIN 3); structural constituent of cell wall 3.6 255345_at AT4G04460 aspartyl protease 3.6 258957_at AT3G01420 ALPHA-D0X1 (ALPHA-DIOXYGENASE 1) 3.3 262930_at AT1G65690 harpin-induced protein-related / HINI-related AT2G15120 disease-resistance pseudogene; sim. to fatty acid elongase 1 3.1 265920_s_at AT2G15220 secretory protein, putative 3 245393_at AT4G16260 glycosyl hydrolase family 17 3 247297_at AT5G64100 peroxidase, putative 3 257654_at AT3G13310 DNAJ heat shock N-terminal domain-containing 3 267238 at AT2G44130 kelch repeat-containing F-box family 2.7 260551_at AT2G43510 ATTI1 (A. thaliana trypsin inhibitor protein 1) 2.7 263228_at AT1G30700 FAD-binding domain-containing protein 2.6 257774_at AT3G29250 oxidoreductase 2.6 259036_at AT3G09220 LAC7 (laccase 7); copper ion binding / oxidoreductase 2.5 257944_at AT3G21850 ASK9 (Arabidopsis SKP1-LIKE 9); ubiquitin-protein ligase 2.5 264005_at AT2G22470 AGP2 (Arabinogalactan-protein 2) 2.4 247059_at AT5G66690 UGT72E2; UDP- or coniferyl alcohol-glucosyltransferase 2.4 254101_at AT4G25000 AMY1/ATAMY1 (ALPHA-AMYLASE-LIKE); alpha-amylase 2.4 260568_at AT2G43570 chitinase, putative 2.3 249375_at AT5G40730 AGP24 (Arabinogalactan protein 24) 2.3 250724_at AT5G06330 harpin-responsive protein, putative (H1N1) 2.3 252958_at AT4G38620 MYB4 (myb domain protein 4); transcription factor 2.3 254396_at AT4G21680 proton-dependent oligopeptide transport (POT) family 2.3 254828_at AT4G12550 AIR1 (Auxin-Induced in Root cultures 1); lipid binding 2.3 258791_at AT3G04720 PR4 (PATHOGENESIS-RELATED 4) 2.2 247333_at AT5G63600 flavonol synthase, putative 2.2 264998_at AT1G67330 similar to ATIG27930.1; contains DUF579 2.1 246302_at AT3G51860 CAX3 (cation exchanger 3); cation:cation antiporter 2.1 253676_at AT4G29570 putative cytidine deaminase / cytidine aminohydrolase 2.1 260039_at AT1G68795 CLE12 (CLAVATA3/ESR-Related 12); receptor binding 2.1 261930_at AT1G22440 alcohol dehydrogenase, putative 2 248062_at AT5G55450 protease inhibitor/seed storage/lipid transfer protein (LTP) AT5G43350 ATPT1 (Phosphate Transporter 1) 2 249152_s_at AT5G43370 APT1/PHT1;2/PHT2 (Phosphate Transporter 2) 63 Table 5.2 (Continued) 2 249777_.at AT5G24210 lipase class 3 2 257697_.at AT3G12700 aspartyl protease 2 264685_.at AT1G65610 ATGH9A2/KOR2 0-glycosyl hydrolase 9A2 2 266884_at AT2G44790 UCC2 (UCLACYANIN 2); copper ion binding 1.9 247312_.at AT5G63970 copine-related 1.9 247327_ at AT5G64120 peroxidase, putative 1.9 249346_.at AT5G40780 LHT1 (Lysine Histidine Transporter 1) 1.9 249767_at AT5G24090 acidic endochitinase (CHIB1) 1.9 251843_.x_at AT3G54590 ATHRGP1; structural constituent of cell wall 1.9 257066_.at AT3G18280 protease inhibitor/seed storage/lipid transfer protein (LTP) 1.9 260887_.at AT1G29160 Dof-type zinc finger domain-containing protein 1.9 266168_.at AT2G38870 protease inhibitor, putative AT4G36988 CPuORF49 (Conserved peptide upstream ORF 49) 1.8 246214_.at AT4G36990 HSF4 (Heat Shock Factor 4); transcription factor 1.8 255904_.at ATI G17860 trypsin and protease inhibitor / Kunitz family protein 1.8 256883_ at AT3G26440 similar to ATI G13000 1.8 260391_.at AT1G74020 SS2 (STRICTOSIDINE SYNTHASE 2) 1.8 262682 .at AT1G75900 family II extracellular lipase 3 (EXL3) 1.8 262942_at AT1G79450 LEM3 (ligand-effect modulator 3) / CDC50 family 1.7 246125_.at AT5G19875 similar to AT2G31940.1 1.7 254234_.at AT4G23680 major latex protein-related / MLP-related 1.7 254314,.at AT4G22470 protease inhibitor/seed storage/lipid transfer protein (LTP) 1.7 258596,.at AT3G04510 similar to LSH1 Light-dependent Short Hypocotyls 1 1.7 265943. at AT2G19570 CDA1 (CYTIDINE DEAMINASE 1) 1.7 267128..at AT2G23620 esterase, putative 1.6 249955. at AT5G18840 sugar transporter, putative 1.6 254040..at AT4G25900 aldose 1-epimerase family protein 1.6 254500. at AT4G20110 vacuolar sorting receptor, putative 1.6 255059. at AT4G09420 disease resistance protein (TIR-NBS class), putative 1.6 258374..at AT3G14360 lipase class 3 family protein 1.6 260408._at AT1G69880 ATH8 thioredoxin H-type 8 1.6 265111. at AT1G62510 protease inhibitor/seed storage/lipid transfer protein (LTP) 1.6 266808’_at AT2G29995 similar to ATI G07175.1 1.5 246620._at AT5G36220 CYP81D1 (cytochrome P450 91 Al); oxygen binding 1.5 262657._at AT1G14210 ribonuclease T2 family 1.4 254818._at AT4G12470 protease inhibitor/seed storage/lipid transfer protein (LTP) 1.4 264708._at AT1G09740 ethylene-responsive protein, putative AT5G25980 TGG2 glucoside glucohydrolase 2; 0-glycosyl hydrolase 0.8 246880._s_at AT5G26000 TGG1 (thioglucoside glucohydrolase 1) 0.8 261711._at AT1G32700 zinc-binding family 0.7 244972. at ATCG00680 CP47, subunit of the photosystem II reaction center. 0.7 245024._at ATCG00120 ATPase alpha subunit, thylakoid membrane 0.7 246031._at AT5G21160 La domain-containing protein / proline-rich 0.7 246270._at AT4G36500 similar to AT2G18210 0.7 246396._at AT1G58180 carbonic anhydrase / carbonate dehydratase family 0.7 249867._at AT5G23020 IMS2/MAM-L/MAM3 (methylthioalkymalate synthase-like) 0.7 249941 _at AT5G22270 similar to AT5G06270.1 0.7 251109._at AT5G01600 ATFER1 (FERRETIN 1); ferric iron binding 64 Table 5.2 (Continued) 0.7 253208_at AT4G34830 binding 0.7 253647_at AT4G29950 microtubule-associated protein 0.7 254256_at AT4G23180 CRK10 (cysteine-rich RLK10); kinase 0.7 254687_at AT4G13770 CYP83A1 (cytochrome P450 83A1); oxygen binding 0.7 254784_at AT4G12720 AtNUDT7; hydrolase/ nucleoside-diphosphatase 0.7 257021_at AT3G19710 BCAT4 MET-oxo-acid or branched-chain aminotransferase 0.7 260726_at AT1G48160 signal recognition particle 19 kDa protein / SRP19, putative 0.7 260969 at AT1G12240 p-FRUCT4/vacuolar invertase; p-fructofuranosidase AT1G48598 CPuORF31 (Conserved peptide upstream ORF 31) 0.7 261309 at AT1G48600 phosphoethanolamine N-methyltransferase 2, put. (NMT2) 0.7 261459_at AT1G21100 0-methyltransferase, putative 0.7 261667_at ATI G18460 lipase 0.7 261951 at AT1G64490 similar to AT5G42060.1; contains DEK C-terminal domain ATI G17280 UBC34; ubiquitin-conjug. enzyme 34; ubiquitin-protein ligase 0.7 262537 s at AT5G50430 UBC33; ubiquitin-conjug. enzyme 33; ubiquitin-protein ligase 0.7 263039_at AT1G23280 MAK16 protein-related 0.7 263443_at AT2G28630 beta-ketoacyl-CoA synthase 0.7 265484_at AT2G15820 pentatricopeptide (PPR) repeat-containing protein 0.7 266106_at AT2G45170 AtATG8e (AUTOPHAGY 8E); microtubule binding 0.6 245119_at AT2G41640 similar to AT3G57380 glycosyltransferase; DUF563 0.6 247867_at AT5G57630 CIPK21 (CBL-interacting protein kinase 21); kinase 0.6 249866_at AT5G23010 MAM1, 2-isopropylmalate synthase 3 0.6 252648_at AT3G44630 put. disease resistance, RPPI-WsB-like/TIR-NBS-LRR class 0.6 255016_at AT4G10120 ATSPS4F; sucrose-phosphate synthase 0.6 255285_at AT4G04630 similar to AT4G21970.1; contains DUF584 0.6 255626_at AT4G00780 meprin and TFtAF homology (MATH ) domain-containing 0.6 259541 at AT1G20650 protein kinase AT1G73600 phosphoethanolamine N-methyltransferase 0.6 259842 at AT1G73602 CPuORF32 (Conserved peptide upstream ORF 32) 0.6 261901_at AT1G80920 J8; heat shock protein binding / unfolded protein binding 0.6 261957_at AT1G64660 ATMGL; catalytic/ methionine gamma-lyase 0.6 267477_at AT2G02710 PAC motif-containing protein 0.5 245777_at AT1G73540 ATNUDT21; Nudix hydrolase homolog 21 0.5 249337_at AT5G41080 glycerophosphoryl diester phosphodiesterase 0.5 249862_at AT5G22920 zinc finger (C3HC4-type RING finger) 0.5 253874_at AT4G27450 similar to AT3G15450.1 0.5 256613_at AT3G29290 EMB2076 (EMBRYO DEFECTIVE 2076) 0.5 260126_at AT1G36370 SHM7 serine/glycine hydroxymethyltransferase 0.5 266313 at AT2G26980 C1PK3 (CBL-interacting protein kinase 3); kinase AT2G32870 meprin and TRAF homology (MATH ) domain-containing 0.5 267644 s at AT2G32880 meprin and TRAF homology (MATH ) domain-containing 0.4 247954_at AT5G56870 BGAL4 (beta-galactosidase 4); beta-galactosidase 0.4 257766_at AT3G23030 IAA2 indoleacetic acid-induced protein 2; transcription factor ATI G71330 ATNAP5 (non-intrinsic ABC protein 5) 0.4 259937_s_at AT3G13080 ATMRP3 (multidrug resistance-associated protein 3) 0.3 250327 at AT5G12050 similar to unnamed protein (Vitis vinifera GB;CA045643.1)
65 A • B C D
n
■ C - ^ ■ ' p - ■ t
0.2% DMSO only 50 ppm BaP in 0.2% DMSO
Figure 5.1 Plants with pink methylobacteria colonizing the rhizosphere, roots, and vasculature in 0.2% DMSO control agar (A) and 50 ppm BaP (C) had thicker leaves and stems than their sterile-cultured counterparts in both DMSO control agar (B) and BaP- containing media (D). Note that sterile plants in (B) and (D) were approximately 2 months old, and control rosettes (B) were chlorotic, with roots becoming nonfunctional.
Plants in (D) appeared to have narrower stems, but this was not a consistent difference.
6 6 CONTROL 50 ppm BaP
Figure 5.2 No clear phenotypic differences were seen between soil-grown control plants
(left) and BaP-grown plants (right). All plants were healthier than plants grown in agar, with larger, thicker leaves and more obvious trichomes. Older plants displayed anthocyanin pigmentation in the stems. No solvent was used for either group, as the acetone was evaporated from both samples before planting.
67 GO Cellular Component, Control
c S T vtosol iu: 4% ^ 2. i%------■ extracellular 4; 2% □ Golal apparatus 0: OTo e g mitochondria 9, 4 3 0 cinucleus 25: 11 ■ other cellular components 24; 10% a other cytoplasmic components 30: 13% CM Other intracellular components 4b: 2 U % :: □ other memuranes 23, 10% ■ plasma membrane 22; 10% e n p la s tid 12: 5 % ^ ■ ribosome 2; 1% □ unknown cellular components 79; 34%
GO Cellular Component, BaP Cgcell wall 27; 1 3 ^ ^ □ c(iloroplast4, 2% □ cytosol 2; 1 % □ ER 3: 1%______C g e x tra c e llu la r 13; 6 % " !: : □ Goigi apparatus 1; U% m m itochondria 4; 2% nucleus 9- 4% < g other cellular components 61: 3 0 % "^ ioinercytopiasmic components 2.s n v \% ■ other intracellular components 23; 11 % < D other m em branes 3b: i /7o ■ piasma memorane 2 /; i i % □ plastid 3; 1% ■ ribosome 1; 0% □ unknown cellular components 51; 25%
Figure 5.3 Comparative distribution of cellular components based on Gene Ontology (GO) annotation of eellular loealizations of genes that were differently expressed in eontrol vs. BaP-exposed plants. Data were compiled using TAIR GO annotation search, functional categorization and download tools, available at: http:/7www./1ra/?/c/op5/.y.org/tools/bulk/go/index.isp. Groups over-represented in controls or BaP plants are circled.
6 8 GO Biological Process, Control □ cell organization and biogenesis 9, 3% developmental processes 11; 4% DNA or RNA metabolism 3: 1 % □ electron transport or energy pathways 7; 3% n nthpr hinlnniral prnrP^^P^ 3Q: 1 2% Other cellular processes 105: 41 OTner metaooiic processes ifjU; 3^ o ■ protein metabolism 26; 10% □ response to abiotic or biotic stimulus 35; 14% ■ response to stress 34: 13% □ cell organization and biogenesis 4; 2% GO Biological Process, BaP ■ developmental processes 9; 4% □ DNA or RNA metabolism 0; 0% □ electron transport or energy pathways 6; 3% [other biological processes 35; 17% I other cellular processes bU; 20% I other metabolic processes 76; 37% I protein metabolism 17: 8%______CTTTrp^^ponse to abiotic or biotic stimulus 39: 19^?T!> cT B response to stress 52; 25% □ signal transduction 8; 4% ■ transcription 7; 3% C^transport 27; 13~^T^ ■ unknown biological processes 50; 24% Figure 5.4 Differential GO annotation of biological processes in control vs. BaP- exposed plants. Processes represented by more gene aimotations in control (down- regulated in BaP/control) or in BaP (up-regulated) are circled. 69 GO Molecular Function, Control CODN^or RNA bindingljr i ^ ■ hydrolase activity 31; 12% ■ kinase activity 20; 8% □ nucleic acid binding 2; 1% nucleotide binding ■ other binding 33; 13% ■ other enzyme activity 32; 13% ■ other molecular functior^ 7: 3% C j ^ o t e i n binding 24; l"o K ir2> □ receptor binding or actMty 3; 1% □ structural molecule activity 1; 0% ■ transcription factor activity 21; 8% rCStransferase activity 39;T b^^I> □ transporter activity 14: 6% CjTunknown molecular functions 80:~3?^> □ DNA or RNA binding 7; 3% GO Molecular Function, BaP ■ hydrolase activity 31; 15% ■ kinase activity 6; 3% □ nucleic acid binding 0; 0% □ nucleotide binding 4; 2% C jo t h e r binding 3 6 ; l 8 ^ [other enzyme activity [5fher molecular functions 12; ■ pimtein binding 10; 5% ^ □ receptor binding or activity 2; 1 % □ structural molecule activity 3; 1 % ■ transcription factor activity 14; 7% □ transferase activity 15; 7%i CPPTransporter activity 19; ■ unknown molecular functions 44; 22% Figure 5.5 Differential GO annotation of molecular functions in control vs. BaP- exposed plants. Functions over-represented in either control or BaP-exposed plants are circled. 70 High expression B BaP Plants □ Control Plants NudtZ MRP3 Myb4 GDE Medium expression Low expression 0.045 -1 0.0025 1 0.04 - 0.035 - 0.002 - 0.03 0.0015 0.025 - 0.02 - 0.001 - 0.015 - 0.01 - 0.0005 0.005 0 NAP GH17 SS DOXl PRX3a Lac7 Figure 5.6 Expression of 10 genes in soil-grown Arabidopsis, measured by qRT-PCR. Bars represent standard error of three or four biological replicates. 71 6 - □ GeneChip (Agar) i= 5 - c □ • qRT-PCR (Soil) o o Q . , (0 4 ffl c o □ ■«« 3 □ 2! Q. □ □ 0)X 2 - ■a 0) N m E 1 1. o c = □ □ 1 1 1 1 \ 1 1 1 1------SS AtNAP PRX3a GH17 Myb4 Nudt7 MRP3 GDE Lac7 D0X1 Figure 5.7 Comparative expression of 10 genes measured in two independent experiments, expressed as the ratio of BaP vs. control plants. In the first experiment, microarrays were used to analyze gene expression of Arabidopsis grown in agar with 50 ppm BaP in 0.4% DMSO, or DMSO only. In the second experiment, Arabidopsis plants were grown for 4 wks in soil with 0 ppm or 50 ppm BaP, and expression of 10 genes was measured by qRT-PCR. 72 5.4 Discussion Since the plants grown in BaP in agar for 4 wks lived longer than controls, it would be useful to determine whether BaP mitigated growth conditions by providing a hydrophobic barrier on the root surfaces, an additional carbon source, or whether BaP actually stimulated genes that resulted in life extension. In animals, BaP has been shown to have an immune stimulatory effect, even enhancing the production of specific antibodies toward unrelated antigens. For example, BaP coadministered with ovalbumin stimulated production of ovalbumin antibodies in mice, while either BaP or ovalbumin alone did not.’®® The authors measured increased secretion of Thl cytokines, Th2 cytokines IL-4 and IL-10, IFN-gamma, and IL-12p70. Burchiel et al. found that diesel exhaust suppressed, but BaP-quinones stimulated, T cell proliferation.’®^ BaP-quinones can be produced enzymatically by peroxidases and cytochrome P450 enzymes, or by photochemical oxidation. Since quinones are among the most commonly reported PAH metabolites found in plants, a parallel phenomenon in plants might explain the increased lifespan of the 4-wk exposed plants. Comparison of plant genes responding to BaP with homologous mammalian genes induced or repressed by BaP may show parallels with immunostimulation, in addition to degradation pathways. Individual genes with differential expression in Arabidopsis in response to BaP in agar and in soil are discussed below. DOXl (At3g01420) Arabidopsis alpha-dioxygenase 1 (DOXl) contains a conserved animal haem peroxidase domain, and has 38% and 36% amino acid similarity to human 73 cyclooxygenases COX-1 and COX-2, respectively. Both of the human COX enzymes are able to activate BaP and its metabolites.’®* COX-1 is constitutively expressed in most cases, although it may be up-regulated by TNF-alpha, TGF-Bl, and ILl- B,’®” and promotes angiogenesis.” ” COX-2 is induced by BaP metabolites.’®* COX-1 and -2 also act on lipid substrates such as arachidonic acid to generate prostaglandins and other potential lipid signaling molecules. DOXl has alpha-dioxygenase activity on a variety of endogenous fatty acids, including linolenic, linoleic, and palmitic acids, and less activity on arachidonic acid. Unlike COX-1 and COX-2, no peroxidase activity was found.’^’ It is not clear if DOXl can oxidize BaP. DOXl can be induced by reactive oxygen or electrophilic species, producing oxygenated lipids involved in defense signaling and protection against cell death.” ’ The resulting 2-hydroperoxide intermediates could be used to produce jasmonate, but there is insufficient evidence to conclude this, as the next two enzymes in the pathway were not up-regulated by 4-wk exposure to BaP. Both animal COX and plant DOX enzymes act in inflammatory response pathways, and can result in oxygenated lipid products with hormone activity (prostanoids and jasmonates, respectively). DOXl may not share the COX enzymes’ ability to promote cancer processes such as angiogenesis and metastasis, but it does inhibit apoptosis. The EGF domain found only in the cyclooxygenases may also contribute to the development of cancer in animals. Lac7 (At3g09220) Laccase 7 (Lac7) is a member of the multicopper oxidase family, which has homology to fungal laccases (29% BLASTp identity with Pleurotus sp. 'Florida' CAA06291.1, Expect = 2e-49). Fungal laccases can degrade PAHs, and P- 74 hydroxycinnamic acids such as p-coumaric and ferulic acids enhance fungal laecase activity to achieve up to 90% degradation of BaP within 48 hours.” ’ Plant laecases aid in lignin polymerization, while fungal laccases act to break lignin down. Although it is not clear whether plant laceases can act on BaP, their participation in lignin formation coupled with the fact that fungal laccases transform BaP into quinones suggests that Lac7 may be at least partly responsible for the incorporation of BaP quinones into lignin which was modeled in vitro with horseradish peroxidase plus coniferyl and vanillyl alcohols.” * PRX3a (At5g64100) Peroxidase 3a (PRX3a) belongs to the class III plant secretory peroxidases, that include horseradish peroxidase. Peroxidases regulate H 2 O2 levels by transferring electrons from H2 O2 to a substrate such as monolignol or possibly BaP, producing radicals either as by-products or defense compounds. Peroxidase activity is involved in defense response, senescence, fruit ripening, cell expansion and cell wall cross-linking. Peroxidases may be involved in auxin catabolism, or may modulate auxin effeets, since production of auxin correlates with higher levels of ROS and leads to wall expansion.” * PRX3a was induced 2.5 fold after 10 d growth on MS with 100 uM arsenate.” * It was co-induced with its tandem neighbors, At5g64110 and At5g64120, in aecordance with previous reports that tandemly arranged peroxidases share expression profiles.'” At5g64110 has 70% amino acid identity, and At5g64120 has 58% identity, to PRX3a, which is consistent with the possibility that these sequences represent tandem duplications. The three genes are identified as eoexpressed in the ATTEDII database, with PRX3a more highly correlated to At5g64110 than to the more downstream At5g64120. In the BaP-exposed plants, At5g64120 up-regulation was closer to that of 75 PRX3a than At5g64110. This may be due to regulated induction specific to At5g64120, as this gene has been reported to be inducible by the allelochemical BOA,"* and involved in respiratory burst associated with fungal defense response."® MRP3 (At3gl3080) This protein has 34% identity and 53% similarity to human MRP 1-4 (also called ABCCl through 4), with an expect value of 0.0. Human MRPl (multidrug-resistant protein 1) is overexpressed in drug-resistant cancer cells,'*** but the mechanisms of up- regulation were only recently elucidated. Single-dose doxorubicin led to acquired drug resistance and up-regulation of a related MRP in breast, ovarian and colon cancer cells 181 after only ten days. The overexpression was traced to weakened HDACl-promoter association, leading to histone hyperacetylation. Arabidopsis MRP3 was induced by four xenobiotics (l-chloro-2,4-dinitrobenzene, metolochlor, primisulfuron, and IRL1803, an experimental herbicide that inhibits histidine synthesis) over the entire length of a time course that included measurements at 12 h, 48 h, and 60 h post-exposure.'*^ Repression of MRP-type transcripts in mammals has been reported in response to various stresses and signals. Repression of human ABCAl transcription is mediated by USFl and USF2 binding to an E-box 147 bp upstream of the transcriptional start site is bound by USFl and USF2.'*^ Another group found that ABCAl repression may be mediated by liver X receptor-nuclear corepressor complex binding.'*'' Estrogen lowered expression of chick MRPl homolog.'*^ It was not clear why MRP3 was down-regulated in BaP-exposed plants, but qRT-PCR confirmed that the transcripts were somewhat lower in the soil experiment. 76 GLf77(At4gl6260) At4g16260 is a member of glyeosyl hydrolase family 17 (GH17) which has only been identified in plants and fungi.'** This family is defined by CAZY, but includes enzymes with endo-l,3-beta-glucosidase (EC:3.2.1.39), lichenase (EC:3.2.1.73), and exo- 1.3-glucanase (EC;3.2.1.58) activity. According to the eFP Browser summary of microarray data, GH17 is induced by osmotic, salt, oxidative, methyl viologen, pathogens, ethylene, drought, UV-B and wounding.'*’ A stress- and cold-induced endo- 1.3-P-glucosidase from tobacco was cryoprotective when incubated with spinach thylakoids.'** The enzyme decreased the permeability of vesicle membranes, reducing solute loading and subsequent vesicle mpture on freezing. Oligosaccharides produced by this enzyme may induce defense response genes, based on evidence from plant pathogen-derived saccharides.'*" There is a growing body of evidence that suggests glycosylation and carbohydrates play many roles in cancer- related processes.'"" A National Cancer Institute (NCI) initiative funded seven Tumor Glycome Laboratories to identify possible glycan markers for cancer.'"' GH17 also contains an RGD tripeptide, that could be involved in interaction with an EGF-containing protein in an integrin-like signaling pathway. Finally, GH17 enzymes can cleave glycosidic bonds between two carbohydrate moieties or between a carbohydrate and a non-carbohydrate. GH17 might therefore be able to de-glycosylate BaP. AtNap (At4g04460) The aspartyl protease, At4g04460, is the HomoIoGene for human Napsin A (E=2e- 65). Human NapA is expressed at high levels in the lung and is considered a marker for primary tumors and metastases of lung adenocarcinoma.'"’ NapA has a C-terminal 77 insertion of four amino acids (DMKS) not found in other napsin homologs, which forms an RGD, the recognition site for integrin binding. At4g04460 has no RGD but contains a KGE (a.a. 252-254), which is a similar recognition site in plants. This KGE aligns exactly with an RGD in cardosin A from Cynara cardunculus (cardoon), which, together with a more C-terminal KGE, was bound by the C2 domain of phospholipase D-alpha.’”® Human phospholipase D2 (PLD2) has close homology to PLD-alpha from cardoon, and has been associated with invasion and metastasis.’”"* In the same study, inactive PLD2 (with a K758R substitution in the catalytic site) inhibited adhesion, invasion and metastasis in lymphoma cells. It is possible that the anticancer effects of inactivated PLD2 are mediated by its C2 domain binding RGD or KGE tripeptides, which are known to be involved in adhesion signaling. PLD2 has also been shown to bind EGFR,’”® which is partially activated by integrin receptors even in the absence of EGF and is required for activation of downstream enzymes involved in proliferation.’”® ^tNap also has high homology to human Cathepsin D (E=8e-58), but lacks homology in the propeptide region which was found to be involved in invasion, metastasis, and proliferation in breast cancer cells.’”’ Cathepsin D activity is linked to anticancer processes when it acts on specific protein targets. In fact, one of the main carcinogenic effects of COX-2 involves Cathepsin D. COX-2 overexpression increases angiogenesis by inhibiting Cathepsin D cleavage of plasminogen to angiostatin.’”^ In Arabidopsis, three aspartyl proteases, including v4tNap, contain a unique plant- specific sequence (PSS) as an approximately 100 amino acid insertion. The insertion includes two motifs called saposin-like type B, 1 and 2. These motifs are not expected to disrupt protease activity, as a chimeric pig protease containing an insert of the plant- 78 specific sequence still showed protease activity.'®® The saposin-like domains appear to be cleaved from the functional aspartyl protease, as saposins A-D are cleaved from a single prosaposin protein. Saposins are non-enzymatic sphingolipid activator proteins (SAP) with important immune functions in mammals. They activate B-glucosidase and other enzymes by transporting sphingolipids or binding to them.’®® Saposin B is required for alpha-galactosylceramide recognition by natural killer T cells (NKT).’®' Recombinant plant Cardosin A saposin-like domain induced vesicle leakage in vitro,^^^ similar to the manner in which homologous NK-lysin forms pores in tumor cells and bacterial membranes.’®’ It is not clear if GH17 saposin-like domains have any function, but they are proposed to target the aspartyl protease to the vacuole.’®'* A«rfr7(At4gl2720) The Nudix motif was first identified in MutT from E. coli, which catalyzes hydrolysis of dGTP to dGMP.’®® Complementation of E. coli with inactive MutT by a human Nudix-type gene showed that the human gene protects against radical-induced mutagenesis by depleting supplies of 8-oxo-dGTP, with a preference for oxo- dGTP>dGTP>dATP.’®® The functions of most Nudix-containing genes are not known. Human Nudt6 is also called GFG, or FGF-AS, as it is encoded by a region containing approximately 400 nucleotides of the 3’ UTR of fibroblast growth factor, in the antisense direction.’®’ Overexpression of GFG suppresses FGFl transcript by RNA interactions,’®* and the 35 kDa cytosolic protein has antiproliferative effects independent of the transcript.’®® Arabidopsis has 24 Nudix hydrolases (nucleoside diphosphates linked to moiety X- type proteins), including nine cytosolic genes that have been shown to have different 79 substrate preferences/’” Recombinant AtNudt? had activity against ADP-ribose and NADH, but not dGTP or 8-oxo-dGTP. ^’” Arabidopsis nudt? mutants showed enhanced defense response and immunity against P. syringae, indicating that Nudt7 functions to suppress pathogen responses/” Another study found that two independent T-insertion mutants were resistant to an oomycete pathogen, dwarfed, and exhibited necrotic lesions/’^ These phenotypes were reversed by a double (nudt?/edsl) mutant, indicating that signaling depends on EDSl. The authors also concluded that Nudt? antagonizes the initiation of cell death, but this contradicts what is known about human GFG. Human GFG suppresses transcript levels of the proliferation and angiogenesis promoter FGF, and delays cell cycle progression/’^ Proliferation, angiogenesis, and uninhibited cell cycle progression are cancer-promoting, while apoptosis generally protects against these processes. AtNudt? expression was induced by inoculation with avirulent more than virulent pathogen, and not at all by mock inoculation,^’'’ indicating that it may help refine non-pathogen defense responses. Expression was also induced by superoxide, and levels of ROS increased in mutants.^’'* Nudix gene responsiveness to PAH is not well-studied, but a putative rat Nudt3 homolog was 0.6x down-regulated by dimethylbenzanthracene (DMBA) + estradiol in a rat microarray study.^’® High FGF expression occurs in gliomas^’® and pituitary tumors,^’” with lower expression of GFG, indicating that low GFG may be expected in most cancers. Myb4 (At4g38620) This gene has homology to the human oncogene c-Myb (e=4xl0'^®), amplified in aggressive primary tumors and advanced metastatic cancer.^’* There are 198 Myb genes in A thaliana^^^ most of which belong to the R2R3 subfamily and are involved in 8 0 controlling plant-specific processes related to phenylpropanoid metabolism.” * Myb4 is an R2R3 Myb that is induced by various oxidative stresses, and mediated by oxylipin products of linolenic acid degradation.” ' Myb4 is a repressor of cinnamate 4- hydroxylase {C4H) and is down-regulated by UV-B. This response is self-destructive, as repressing C4H would result in a decrease in photoprotective sinapate esters.’” However, normal UV response increases sinapate esters, so the authors theorize the existence of a de-repression mechanism as well as possible competitive binding by different Myb proteins to fine-tune responses. In 4-wk BaP exposed plants, neither C4H nor the related ferulate 5-hydroxylase {F5H) that catalyzes the rate-limiting step of syringyl lignin,” * displayed significantly altered expression. One of the three treated biological replicates analyzed by microarray showed different regulation of the flavonoid pathway across many genes; mostly these genes were called ‘increased’ in this sample, with no changes in the others. This likely indicates that one of the pooled plants in this sample had a stronger defense response. 55(Atlg74010) Transcription of two tandemly arranged strictosidine synthase genes (Atlg74010 and Atlg74120) increased in response to BaP. Although strictosidine synthase genes are known for the alkaloids they produce in higher plants, homologs exist in C. elegans and human brain.HomoloGenes include Drosophila hemomucin (implicated in immune response)” * and human adipoeyte membrane-associated protein (APMAP), induced during adipoeyte differentiation.” * It is not clear if Arabidopsis is capable of making complex alkaloids,” ’ so it is possible that these two genes may serve some additional defense response purpose, similar to hemomucin or APMAP. Products of plant 81 strictosidine synthases are expected to have defense functions, and strictosidine had antibiotic activity in Catharanthus roseus. Homologs of SS are responsible for production of the antineoplastic compounds vincristine and vinblastine, so the up- regulation of these genes in Arabidopsis carcinogen response may indicate an adaptive response. Metabolite profiling of Arabidopsis has not so far produced evidence of such complex alkaloids in this plant, but this particular inducer has not been used. It would be interesting to look for new compounds that might be produced in Arabidopsis only under carcinogen or PAH stress. In the field of pharmaceuticals, PAH compounds less hazardous than BaP may be worth investigating, to see if they can induce, enhance, or alter the form of alkaloid production in plants. As anticipated, microarray screening of plant BaP response revealed gene expression changes in homologs to genes from other organisms that are known to be activated or repressed by BaP. A homolog to mammalian COX-2 may not retain the ability to oxidize BaP, but it appears to act in similar ways against fatty acids and cell death. In addition, a member of an enzyme family that epitomizes fungal BaP degradation, Lac7, was induced, as was a plant secretory peroxidase, which was expected to be involved in plant degradation of BaP. Mrp2 and Nudt7 are homologs for human genes involved in xenobiotic transport and suppression of proliferation and angiogenesis, respectively. These were both down-regulated in Arabidopsis-BsP interaction, and it is not clear if the genes share the same functions in animal BaP response. Additional genes related to plant-specific processes were induced as well. GH17 is a member of plant and fungal gene family which can hydrolyze cellulose. Myb4 probably shares an ancestor with viral and cellular Myb genes involved in cancer 82 signaling, but has so far been associated with repression of the plant phenylpropanoid pathway. Strictosidine synthases are only known to assist in alkaloid production in plants, but homologs have been found in mammals. Up-regulation of these enzymes in Arabidopsis response to BaP has not been previously reported. A homolog of a known marker for lung cancer was up-regulated in Arabidopsis, but this gene (AtNap) contains a plant-specific insert that appears to have a separate and not exclusive activity of its own. While plant research can advance by drawing certain parallels with better-known animal-BaP responses, it appears that plants have adapted some unique and possibly more complex strategies. The tumor-targeting tripeptide RGD was present in GH17, and in AtNap the RGD from human Nap A was replaced by the more common plant tripeptide, KGE. Both RGD and KGE are components of vigorous signaling pathways related to adhesion, and RGD has been proposed as a tumor marker and vaccine candidate. Elucidation of a basal function for these tripeptides in plants may help assess potential interactions with non-mmor tissues as researchers try to develop a tripeptide cancer vaccine. 83 CHAPTER 6 Expression Analysis of Plants Grown in BaP for 24 hours 6.1 Introduction In order to investigate short-term gene response to BaP, a 24-hour exposure time was used. In animals or cell lines, gene expression changes in response to BaP are often measured between one and 24 hours after exposure.” "’”" Enzyme induction varies widely with culture conditions and exposure times, and has been noted to decline with BaP exposure time in animal cell lines.” ' For example, H4IIE rat hepatoma cells exposed to BaP had increased ethoxyresomfin-O-deethylase activity (EROD) after 24 h,” ’ but not after 72 h.’** Since the uptake rate of BaP by plants is unknown, and because both initial reaction and actual interaction with the BaP are crucial, a 24 h time frame was used. This period should distinguish between early interactions with the BaP and the potential noise of extensive signaling and crosstalk. For microarray experiments, plants were grown to the mature rosette stage in sterile sand, using DMSO to disperse BaP into the liquid media. The first focus of the microarrays was to find alterations in gene expression that might parallel expression changes in animals. More expression data is available on the carcinogenic effects of BaP than on the mechanisms by which it is phytoremediated. Additional gene changes that might explain plant-specific responses to BaP were also of interest. To replicate the short-term exposure, and for application to phytoremediation, the experiment was repeated with plants grown in soil. DMSO was used to disperse the BaP without disturbing the plant roots. Since soil particles adsorb BaP,” '' it might be less bioavailable, and the amplitude of gene responses might decrease. A study on mice 84 found that although intraperitoneal BaP elicited a rapid 14-fold increase in EROD activity, exposure of the mice to soil spiked with BaP in DMSO resulted in minimal uptake and no significant enzyme induction.^®® Specific genes in the soil-grown plants were analyzed by qRT-PCR, to test this exposure method and also to corroborate the microarray data from sand-grown plants. The different experimental conditions (soil V5. sand, open air vs. sterile culture jars, low v^. high humidity) made this a risky proposition, but if successful it would be more likely to identify BaP-specific gene responses instead of incidental effects. Additional validation of microarray results was provided by comparison with other expression studies, and correspondence with coexpression maps built from meta-analysis of the vast amounts of publicly available microarray data. 6.2 Methods 6.2.1 Plant growth conditions in sand Plant culture for microarray analysis was the same as above (Chapter 5), except plants were grown in sand and not exposed to BaP until 4 weeks after germination. Seeds were surface-sterilized as before, plated on MS agar plates, stratified 3 d at 4°C, and transplanted 12 d after germination into tissue culture jars. Each jar contained 70 g of sterile Ottawa sand (Sigma, St. Louis, MO), washed 3x in ddH20, baked for 5 h at 550°C, capped and autoclaved, and 15 mL of Va strength Murashige-Skoog basal salts -i- Gamborg’s B5 vitamins (Sigma), 0.5% sucrose (w/v), pH=5.9 added. Higher MS concentrations produced stress symptoms in all plants. Each jar was spiked 27 d after germination by adding 1 mL of new liquid MS containing either 60 pL DMSO, 60 pL 85 H2O, or 750 pg BaP in 60 pL DMSO. The additions were performed in a sterile hood with a sterile 5 mL syringe fitted with an 0.2 pm filter (RC431215, Coming, NY) and mixed in by drawing liquid media in and out of a sterile 1 mL pipet tip lOx, taking care not to contact the leaves. The final BaP concentration was 50 pg/mL (w/v) in 0.4% DMSO (v/v). Light level was at 90 pE m’ s ’ for 8:16 h day/night, and temperature was maintained between 22° and 24° C. 6.2.2 Affymetrix ATHl GeneChip analysis of plants grown in sand Shoot tissue was harvested 24 h after BaP exposure, flash-frozen in liquid nitrogen, and processed as before for microarray analysis (Ch. 5, Methods). Three biological replicates of BaP and DMSO-only controls were analyzed, plus two no-solvent controls. Each sample consisted of at least six pooled rosettes. Order of harvest was (1) DMSO controls, (2) no-solvent controls, (3) BaP-exposed plants. After statistical analysis, interesting genes were identified for further investigation. 6.2.3 Repeat of experiment in soil For qRT-PCR analysis, seeds were surface-sterilized as before, stratified 3 d at 4° C, and pipetted onto soil that had been sterilized by autoclaving at 120°C for 60 min (four times over 4 days, to remove soil bacteria) in 400 mL glass beakers. Plants were grown under short-day (8 h light) fluorescent lighting. Soil was not spiked with BaP until plants were full-grown (about 4 wks after imbibition). Soil was spiked with 2 mg BaP in 160 pL DMSO, dispersed in 40 mL of water, for a final DMSO concentration of 0.4% (v/v) and BaP at 50 mg/L (w/v). Controls were spiked with 160 pL DMSO in 40 mL water, and no solvent controls were simply wetted with 40 mL water. Solutions were dispensed with a glass pipet, so that liquid did not contact the leaves directly. Plants were harvested 8 6 as before at growth stage 3.9, to minimize differences between the two groups due to developmental changes. Plants were also harvested at additional exposure times, between one hour and 3 days after addition of BaP. Plants were again harvested in the order of spiking (DMSO—no solvent—BaP), and to avoid time-of-day effects, samples were processed quickly so that all treatments were harvested within 10 min of each other. 6.2.4 Quantitative RT-PCR of plants grown in soil Primers were designed using Invitrogen OligoPerfect™ Designer, IDT Oligo Analyzer (Integrated DNA Technologies), mFOLD,” ® and Universal Probe Library (Roche). All primers were checked for cross-hybridization using BLASTn (NCBl). RNA was isolated by Qiagen RNeasy Plant kit and DNase-digested with Roche DNase I. After DNase reaction components were removed using a Qiagen RNeasy column, RNA was quantified using a Thermo Scientific NanoDrop™ 1000 Spectrophotometer, and reverse-transcribed with Bio-Rad iScript cDNA Synthesis kit. Quality and purity of cDNA, and primer design, were evaluated with 3.5% agarose gel electrophoresis of PCR products (Eppendorf) in 0.5x TBE. Quantitative RT-PCR was performed with 20 pL rxns using Bio-Rad iQ SYBR Green Supermix on an IQ5 cycler (Bio-Rad). Reactions included 125 to 500 nM primers and 20 ng cDNA or the equivalent negative control (iScript reaction without reverse transcriptase) to test for genomic DNA (gDNA) contamination or non-specific amplification. Data were considered acceptable if the difference in threshold crossing values (delta-Ct, or dCt) between each cDNA and its corresponding negative control was at least 10 cycles. Five potential reference genes were selected based on the 4 wk and 24 h microarray data and reports of low genotoxic or abiotic stress responses (checked with eFP Browser at the Bio-Array Resource for 87 Arabidopsis Functional Genomics)/^” Reference gene amplification results were reviewed using GeNORM/^* and data were normalized to Tubulin 8 (At5g23860). Background subtracted signals (raw data) were analyzed by PCR Miner (Stanford University, Stanford, CA)^^” and compared in Microsoft Excel. Quantitative RT-PCR products were checked for nonspecific amplification by melt curve analysis and by electrophoresis on a 3.5% agarose gel in 0.5x TBE. The full list of primer sequences, melting temperatures, and qRT-PCR parameters are given in Appendix E. 6.2.5 Data analysis and bioinformatics As in Chapter 5, with additional analyses of genes from both time-points. 6.3 Results 6.3.1 General observations Sand-grown plants were small in the enclosed, sterile, semi-hydroponic conditions. The spiking had to be conducted in a sterile biological/chemical hood, so plants were exposed briefly to temperatures of about 26° C as they were transported to another building. By the next day, the BaP-exposed plants had visible anthocyanin pigmentation. Plant phenotypes were more variable when plants were grown in soil. All plants were healthier than enclosed plants grown in sterile agar, developing larger, thicker leaves with more obvious trichomes. These observations were consistent with expectations, since sterile growth conditions limit nutrient availability and gas exchange, and most importantly, prevent beneficial interactions with endosymbiotic and soil microorganisms. 6.3.2 Microarray analysis of 24 h gene expression in sand-grown plants GeneChip analysis by MAS 5.0 and Data Mining Tool identified 28 probe sets as ‘Increased’ following BaP treatment, and 25 probe sets as ‘Decreased’, in at least 8 of 9 comparisons (Tables 6.1 and 6.2). The full list of differentially expressed genes is given in Appendix D. 6.3.3 Gene expression changes after 24 h in soil-grown plants by qRT-PCR The qRT-PCR results are presented in Figure 6.1. It was not possible to completely remove gDNA from all RNA samples, so primers that spanned introns were used wherever possible. Results were evaluated along with dCt values, and amplification of nonspecific products was checked by agarose gel electrophoresis. In a very few instances, as when a gene contained no introns, or expression levels were extremely low, it was not possible to measure expression differences reliably. 6.3.4 Comparison of qRT-PCR and microarray results Expression ratios of genes measured by qRT-PCR of soil-grown plants were plotted against GeneChip results from the sterile agar experiment (Figure 6.2). The qRT-PCR results showed striking agreement with the GeneChip results, even though the experimental conditions were quite different. 6.3.5 Promoter analysis of genes identified by microarray Databases of cis elements remain incomplete, and many families of transcription factors have only a general conserved binding sequence, proposed on the basis of a single family member’s interaction, or no suggested binding elements at all.’"** For this reason, and to maximize data prospecting related to BaP response, transcription factor motifs from other eukaryotes were considered. Predicted gene regulation was based upon a 89 search for short sequence motifs that were over- or underrepresented in microarray data, compared with the occurrence in the genome as a whole and suggest response pathways operating in the experiment. Motifs over-represented in promoters of genes up-regulated by BaP include EE, DNA damage-inducible elements, E box, G box, C box, and 3 conserved WRKY elements. Over-represented down-regulated motifs included Myb, bZIP/APl, AMLl, E box, GBLE, NAC and TATA boxes. Motif occurrence in 24 h and 4 wk up- or down-regulated gene sets are shown in Table 6.3. 6.3.6 Intersection of BaP and cold stress response Many genes originally identified and armotated as cold responsive were up- regulated in 24 h BaP-exposed plants. As some of these responses are mediated by ABA and/or represent common stress pathways, this is not too surprising. Figure 6.3 outlines a common stress signaling pathway mediated by two Drought-Responsive Element Binding (DREB) transcription factors. Data from the eFP Browser was used to show the different effects of abiotic stresses on DREB 1A and DREB2A expression levels after 24 hrs exposure to the stressor. DRE sequences in gene promoters are recognized by DREB 1A and DREB2A in response to various abiotic stresses, and activate transcription of the genes. Both of these transcription factors were increased in 24-h BaP microarrays by 1.9x and 1.6x, respectively, and the prevalence of the promoter sequences increased for genes up-regulated by BaP. Table 6.4 examines genes identified by microarray as up- regulated in 24-h BaP response, which contain drought-responsive elements (DREs) within 1 kb upstream of the start codon. Because of its chemical properties, BaP is likely to elicit responses similar to oxidative, UV-B, and genotoxic (represented in eFP Browser by experiments using Bleomycin and Mitomycin C) stresses. However, for these DRE- 90 containing and probable stress-regulated genes, there are significant differences. Note that C0R15A is down-regulated in these three stresses, and none of the stresses elicit up- regulation of RD29A, GRP7, or the small hydrophobic protein (all of which were increased by BaP in both sand and soil experiments). Four other genes (ERD3, APRR3, C0R15B, At5g23410) are also upregulated by cold and BaP, but not by oxidative, UV-B, or genotoxic stress. In the up-regulation of ACA8, At5g50450, RPS27aA, COLIO, and At2g22450, BaP response is only similar to cold stress, with other stresses not inducing these genes significantly. Drought and heat stress bear almost no similarity to BaP stress at 24 h post-exposure. 91 Table 6.1 Twenty-eight probe sets that increased in the 24 h microarray results. Genes called ‘increased’ by the Affymetrix Data Mining Tool (DMT) in at least eight of nine comparisons were included, with genes selected for qRT-PCR analysis in bold. Note: probe sets may target more than one gene locus. Locus ID Annotation AT5G57110 autoinhibited calcium-transporting ATPase, ACA8 AT5G52310 cold/desiccation-responsive protein RD29A/COR78 AT5G48250 zinc finger (B-box type), similar to CONSTANS homologs AT5G42900 unknown expressed protein, similar to AT4G33980 AT5G23240 DNAJ heat shock N-terminal domain-containing protein AT3G59350 serine/threonine protein kinase, putative AT4G34120 LEJ1 (LOSS OF THE TIMING OF ET AND JA BIOSYNTHESIS 1) AT4G33980 unknown expressed protein AT1G07050 CONSTANS-like protein-related, similar to C0L15 AT1G68050 FKF1/AD03, E3 ubiqultin llgase SCF complex F-box subunit, AT5G42730 AT5G23410 2 loci similar to FKF1 AT1G67970 heat shock transcription factor HSFA8 AT2G42530 cold-responslve protein COR15B AT4G17490 ethylene response factor ATERF-6 AT4G16146 similar to AT1G69510 AT4G37260 myb family transcription factor MYB73 AT5G61380 TOCl (TIMING OF CAB EXPRESSION 1) transcription regulator AT5G61600 ethylene-responsive element-binding family protein AT5G60100 APRR3 (Pseudo-Response Regulator 3); transcription regulator AT5G57220 CYP81F2 cytochrome P450 AT5G50450 zinc finger (MYND type) family protein AT3G55450 protein kinase, putative, similar to APK1B AT4G34950 nodulin family protein AT4G33985 similar to AT2G15590 AT4G24570 mitochondrial substrate carrier family protein AT4G12280 copper amine oxidase AT4G12290 AT1G27730 C2H2 type salt-tolerance zinc finger, STZ/ZAT10 AT1G05560 UGT1 (UDP-glucosyl transferase 75B1) AT2G38470 WRKY 33 transcription factor 92 Table 6.2 Twenty-nine genes represented by 25 probe sets that decreased in the 24 h microarray results. Genes called ‘decreased’ by the Affymetrix Data Mining Tool (DMT) in at least eight of nine comparisons were included, with genes selected for qRT- PCR analysis in bold. Locus ID Annotation AT1G13650 similar to 18S pre-ribosomal assembly protein gar2-related AT2G03810 ATIG 18620 similar to unknown protein AT1G74160 AT1G55960 similar to unknown protein AT3G13062; Lipid-binding START domain AT1G64500 glutaredoxin family protein AT1G69160 unknown protein AT1G69530 ATEXPA1 (A. thaliana EXPANSIN A l) AT1G74670 gibberellin-responsive protein, putative AT2G04039 similar to unnamed protein AT2G30520 RPT2 (ROOT PHOTOTROPISM 2); protein binding AT2G40205 AT3G08520 603 ribosomal protein L41 AT3G11120 (probe set targets 4 loci) AT3G56020 AT2G40610 ATEXPA8 (A. thaliana EXPANSIN A8) AT2G41250 haloacid dehalogenase-like hydrolase family AT2G46670, pseudo-response regulator/T0C1-like protein, putative AT2G46790 pseudo-response regulator APRR9 AT2G46830 CCA1 (CIRCADIAN CLOCK ASSOCIATED 1); transcription factor AT3G02380 C0L2 (CONSTANS-LIKE 2); transcription factor/ zinc ion binding AT3G05900 neurofilament protein-related AT3G17510 CIPK1 (CBL-INTERACTING PROTEIN KINASE 1); kinase AT3G47340 ASN1 (DARK INDUCIBLE 6) AT3G57040 ARR9 (RESPONSE REACTOR 4); transcription regulator AT4G38860 auxin-responsive protein, putative AT5G06980 similar to unknown protein AT3G12320.1 AT5G15850 COLI (CONSTANS-LIKE 1); transcription factor/ zinc ion binding AT5G35970 DNA-binding protein, putative AT5G47700 60S acidic ribosomal protein PI (RPP1C) AT5G57345 similar to unknown 93 Table 6.3 Frequency of promoter motifs in 143 genes that were up-regulated and 100 genes that were down-regulated by BaP at 24 h, and 80 genes up-regulated and 54 genes down-regulated at 4 wks, compared with expected frequency (% in genome). Over represented motifs indicated in bold. 24 h 4w k % % % % genomic kb MOTIF IDENTITY upreg downreg upreg downreg % 11% 14% 15% 15% 11% 1 TGA[C/G]TCA Bzip/API 26% 38% 33% 35% 30% 3 TGA[C/G]TCA Bzip/API 53% 41% 50% 49% 38% 1 AAAAGTA DNA damage 2% 5% 6% 0% 4% 1 TTCCAGAA DNA damage 32% 26% 23% 27% 27% 1 AAAGTTG DNA damage 27% 23% 25% 29% 25% 1 TTTCAGA DNA damage 4% 3% 0% 0% 3% 1 TGTACGG DNA damage 0% 2% 1% 2% 2% 1 GCATCGC DNA damage 36% 12% 9% 11% 7% 1 AAAATATCT EE 45% 51% 36% 42% 38% 1 TGTGGT AML1 9% 15% 8% 9% 14% 1 TGCGGT AMLI-alt. 27% 21% 13% 18% 15% 1 CACGTG E box 42% 50% 34% 45% 36% 1 CATGTG NAC 8% 4% 4% 7% 5% 1 AGGGATG NAC 4% 1% 4% 0% 2% 1 CCACGTGG G BOX 5% 5% 4% 0% 3% 3 CCACGTGG G BOX 7% 8% 1% 2% 3% 1 TGACGTGGGBLE 11% 16% 6% 5% 7% 3 TGACGTGGGBLE 3% 0% 0% 0% 1% 1 TGACGTCA C BOX 5% 1% 0% 4% 3% 3 TGACGTCA C BOX 8% 0% 9% 13% 6% 0.5 TnGCGTG XRE 9% 8% 13% 20% 11% 1 TnGCGTG XRE 30% 29% 21% 33% 29% 3 TnGCGTG XRE 1% 1% 0 4% 0.3% 3 GACACGTATA MYC2 81% 83% 85% 80% 70% 0.5 TATAWAW TATA box 43% 31% 39% 44% 35% 1 TTGACC WRKY 61% 49% 63% 38% 51% 1 TTGACTWRKY 67% 49% 61% 62% 58% 1 1 1 1GAC WRKY 36% 63% 43% 55% 43% 1 TATCCA MYB 94 Table 6.4 BaP-upregulated genes containing DREs in 1 kb upstream are regulated differently by other abiotie stresses. The second column lists the fold changes from 24-h BaP microarrays; fold changes for other 24-h abiotic stresses archived in the eFP Browser are color-coded according to the key below. Fold Change >50 >10 >2 0.5 >0.2 Genes in bold were also up-regulated in soil-grown plants, as measured by qRT-PCR. Gene BaP Cold Osmotic Oxidative UVB Genotoxic Wound Drought Heat STZ 4.7 6.0 17 9.1 2.9 2.1 11 1.9 0.7 1.2 RD29A 4.4 122 50 1.2 47 0.9 1.2 0.8 1.2 1.0 COL10 4.1 L 53 1.1 1.0 1.1 1.4 1.2 1.4 1.2 1.1 At2g40000 3.9 0.7 1.1 1.5 0.4 1.5 1.9 1.7 0.8 0.6 CYP81F2 3.8 23 52 91 0.6 9.2 17 11 0.7 0.9 At4g27280 3.8 0.4 2.2 3.4 1.8 1.5 4.9 1.7 0.8 0.9 At3g59350 3.7 33 1.7 2.1 1.6 1.2 0.7 1.3 1.0 0.9 At5g50450 3.6 1.6 0.9 1.0 0.8 1.1 1.0 1.0 1.3 0.8 At1g51090 3.6 893 16 3.8 20 2.1 1.3 1.9 2.0 2.4 ACA8 3.4 53 1.1 0.9 1.6 1.0 0.8 1.1 0.9 0.7 KIN1, KIN2 3.3 41 23 1.8 26 0.6 0.8 1.2 1.5 1.2 [At4g 12280] 3.2 5.8 8.5 1.6 5.9 4.7 1.9 2.1 1.1 1.3 ZAT6 3.1 21 19 7.8 4.9 1.1 1.4 1.3 0.9 0.6 PDF1.2 3 1.8 6.1 5.5 0.7 46 4.5 29 11 1.8 At4g30650 3 83 1.0 0.8 5.4 0.6 1.0 1.1 1.2 1.1 [At5g23410] 2.9 2.8 3.8 0.9 1.2 1.0 1.1 1.0 0.9 1.2 APRR3 2.9 8.3 2.1 0.9 1.7 0.9 0.7 1.4 0.9 1.5 MYB51 2.8 5.0 1.9 0.7 2.3 1.4 1.5 1.0 0.7 C0L13 2.6 80 15 16 8.4 3.2 1.5 1.5 1.1 2.5 C0R15B 2.5 160 3.8 1.4 12 r 0.9 1.2 1.1 2.2 1.1 [At4g27560] 2.4 62 1.4 1.4 4.0 1.3 1.3 0.9 1.1 1.0 NHL3 2.4 3.0 2.4 2.2 2.3 2.2 2.9 1.3 1.4 0.9 At5g39020 2.4 0.6 2.7 1.4 0.4 2.0 1.5 2.1 1.2 1.1 At4g36010 2.4 50 1.7 2.7 2.5 1.2 1.4 1.2 1.7 1.2 At5g46710 2.3 9.3 0.8 6.8 1.2 2.0 3.0 1.5 1.2 1.0 SIB1 2.3 2.6 9.7 8.5 2.3 5.7 4.8 1.7 2.3 0.9 GSTZ1 2.2 0.7 12 2.1 5.8 2.3 2.4 2.2 1.5 1.1 TAG1 2.2 23 0.9 2.3 0.9 1.2 0.8 1.3 1.4 0.9 0PR1, 0PR2 2.1 0.3 1.4 6.7 0.9 1.4 1.6 3.0 1.3 1.1 [DOGT1] 2.1 2.0 5.7 1.2 2.6 2.1 1.4 0.8 1.0 RPS27aA 2.1 1.5 0.3 1.2 0.5 0.9 0.8 0.9 0.7 0.7 C0R15A 2.1 85 35 0.5 30 0.2 0.4 0.6 0.9 1.0 At4g36500 2.1 3.4 1.3 2.6 0 . 5 r 1 .3 1.8 1.3 1.2 1.2 GRP7 2 98 0.7 1.5 1.0 1.2 1.1 0.9 1.2 1.1 ERD3 2 9.5 0.2 1.1 1.3 1.3 1.5 1.8 1.2 1.2 At2g22450 2 13 1.6 1.0 1.6 1.4 1.1 1.7 0.9 1.2 At3g16530 0.5 1.8 7.9 2.6 4.9 4.3 1.0 1.2 95 Medium expression 6 - 5 c o □ BaP (0(/) 4 - (D Q.u. ■ DMSO Control X 0) 3 - ■D0) .N "to E 2 - o c 1 - llin■ rL rii . r^T_ 1ilBCAT3 UGT1 E4* VAMP E5 E1 T0C1* TAZ FKF DIB P450* High expression Low expression 0.035 120 - 0.030 c 100 - o (/> 0.025 (/) 80 2 Q. 0,020 Figure 6.1 Specific gene expression by qRT-PCR in 24 h soil-grown plants, as the mean of three biological replicate samples, except where one control sample showed interference from high gDNA (*). Bars represent SEM. 96 35 1 ■ GeneChip c • qRT-PCR o 30 v> w 25 - Q. X E 15 - T3 0 _yN 10 - TO E o 5 - c ■ I 2 • w • • 0 “ I 1-----1-----1---- 1---- 1-----1-----1-----1---- 1-----1-----1---- 1---- 1-----1-----1---- 1----■ Figure 6.2 Gene expression in sand-grown plants as measured on ATHl GeneChip, compared with 19 genes measured by qRT-PCR in a subsequent experiment where the plants were grown in soil. All data points represent mean fold change (BaP/control) of 3 biological replicates per treatment, except those marked *, for which one qRT-PCR sample had interference from gDNA. Expression values for qRT-PCR were normalized to Tubulin 8 (At5g23860). 97 Cold > Osmotic > Oxidative Cold >Salt > Osmotic >Drought >Ge|wtpxic 10. 8x 6 . 2k 5. 9x 42 . 4x 4 . 6k 3. 8x 2. 1x 1. 6x —I \-nREFOA -CD-DREBIA 1 9k cJiREBiX^ H a c c g a c n a Tg? ^ - ACCGACNT - I GCCGACFTK 1.5x C------9 l.dx s 2 .3x f U ^ ------RD29A (4.4x) RD29A STZ/ZATIO 4x At2g40000 3.9x (4.4x) At5g48250 At4g27280 3.8x CYP81F2 3.8x 4.1x Atlg51090 3 . 6k At3g59350 3.7x KINl &2 3.3x At5g04340 3 . 3k At5g50450 3.6x At4gl2280 3.2x At4g30650 2.8x ACA8 3.4x AT2G47890 2.6x C0R15B C0R15B (2.5x) PDF1.2 3x (2.5x) At4g36010 (2.5x) APRR3 2.9x At4g36010 (2.5x) SIBl (2.3x) SIBl (2.3x) Myb 51 2.8x At4g36500 2. Ix NHL 3 2.4x TAGl 2 2x AT3G16530 C0R15A C2.1x) GSTZl 2.2x 2x C0R15A (2.1X) RD17 1.9x ERD7 RPS27aA 2. Ix (1.9x) DOGTl 2 Ix COR414-TM1 1 7x LEAH OPRl & 2 2. Ix 1.6x GRP7 2x At3g53990 1.5x MT2A ERD3 2x 1.4x ERD7 (1 9x) Atlg69870 1 6x At2g02100 1 6x Figure 6.3 Microarray results for 24-h BaP overlaid on map of abiotic stresses mediated by two DRE-binding transcription factors (following schematic of Sakuma, et al. 2006).*” Abiotic stresses that induce each DREB factor after 24 h are listed at the top, with corresponding fold changes underneath (data from eFP Browser). Numbers in red are fold changes in response to 24-h BaP, as measured by microarray. Fold changes in parentheses reflect genes with more than one type of DRE in promoter, and are listed in both columns. Bold red is used to show overrepresentation frequency of DRE motifs in 1 kb upstream of genes increased after 24-h BaP, compared with whole genome. 98 6.4 Discussion In mammals, the signaling response to BaP exposure begins with binding of BaP to the aryl hydrocarbon receptor, AhR.’"”’’*’ BaP-bound AhR then transloeates to the nucleus where it dimerizes with the aryl hydrocarbon receptor nuclear translocator (Amt), and the complex activates transcription of nuclear genes with xenobiotic response elements (XRE) in their promoter regions. No specific homologs of AhR have been identified in plants, but other members of the Per-Amt-Sim family, for which the PAS domain was named, exist in plants. The genes in the PAS family have a wide range of functions, including circadian clock regulation, xenobiotic response, development, and cell lineage control. Besides the PAS domain, some members of this family have a basic- helix-loop-helix domain (bHLH), potentially involved in transcriptional specificity and/or dimerization.’"** The PAS domain is also the site of BaP binding to AhR. A recent report identified genes that were down-regulated in pancreatie cancer as homologs of circadian genes in Drosophila, confirming other evidence that dismption of circadian rhythms is coincident with cancer.’"" The implication of this connection is that BaP may bind to the PAS domain in cireadian genes. Experiments with AhR knockout mice showed that the AhR ligand, dioxin, altered the light responsive circadian rhythms and the expression levels of the two core clock genes Perl and BMALI?^^ The PAS-containing clock signaling gene, FKFl, was up-regulated in Arabidopsis after 24 h of BaP exposure. The xenobiotic response element (XRE) was found by motif analysis of upstream promoter regions. In 4-wk treated plants, XRE motifs were present upstream of down- regulated genes at a level almost twice the genomic level. This pattern held whether the upstream region was 500 bases or 1000 bases from the start codon. This suggests a BaP- 99 mediated mechanism, but without an AhR homolog. While generally considered an enhancer element in BaP or dioxin response mediated by AhR, an XRE has been shown to be required for dioxin-induced down-regulation of prostaglandin endoperoxide G/H synthase-2 gene in rat thymocytes.’"’® It is also present in the promoter region of CYP2C11, down-regulated in rats exposed to dioxin.’"” An animal system that lacks a functional AhR would make a better comparison for plant response. In a rat comeal epithelium culture lacking AhR protein, researchers demonstrated that non-AhR-bound ARNT, HNFl, and HNF4 bound to the XRE region and mediated constitutive induction of ALDH3?^^ Mutations in the XRE region abolished binding and gene activation. The presence of an XRE in disproportionate numbers of genes down-regulated by 4-wk BaP exposure (Table 6.3) could reflect interference with XRE-mediated constitutive transcription. Specifically, BaP may bind to a PAS or PAC-containing protein, favoring formation of a dimer with a protein that normally would bind the XRE for constitutive activation. Thus the genes with XRE in their promoters would appear down-regulated, because BaP dismpted their basal transcription. Such crosstalk is very common, as AhR, ARNT, HNFl, HNF4, and many others can bind the XRE in multiple homo- or heterodimer configurations. It is also possible that the normal activation of these down-regulated genes is via endogenous plant compounds such as those studied in animal systems for their ability to interfere with AhR pathway activation. Epigallocatechin gallate (EGCG) has been shown’"’® to stabilize a form of AhR complex which is then unable to bind the XRE motif or ARNT protein. In a system such as Arabidopsis that may lack an AhR, EGCG may interact similarly with an analogous protein. This interaction could easily interfere with constitutive regulation of a 1 0 0 large set of genes. This possibility is supported by the observation that genes producing flavonoid compounds like EGCG are up-regulated in BaP-exposed plants (At5g63600 and At5gl3930, Appendix C). The determining factor in plant BaP response may start upstream of the regulation of EGCG, and define one of the basic differences between plant and animal BaP response. Correlations between the 24 h response data and known circadian and cold response pathways were found. Although the microarray data may have been skewed by the later harvest of BaP-exposed plants to that of the DMSO control plants, the subsequent experiment using plants grown in soil exercised good control over time of harvest, and the qRT-PCR results verified that FKFl and TOCl were up-regulated in these plants as well. These two genes are central to the circadian clock in Arabidopsis?^^ Genes responding to 24 h cold treatment overlapped extensively with expression profiles of 3 h osmotic and 0.5 h drought treatments.” ' Laboratory studies have shown that short day light regimes elicit enhanced immune response in animals (c/ Nelson et al. 1996),” ’ and that the immunity is mediated by melatonin.” ’ This is presumably an adaptive advantage because short days coincide with the onset of winter, with accompanying cold temperatures and often reductions in food supplies. In plants, photoperiod responses, circadian rhythm, and cold signaling are interrelated.” '' As light is fundamental for their survival, plants that develop efficient mechanisms for sensing light fluctuations will maximize fitness. Since darkness is accompanied by a decrease in temperature, it is reasonable to expect interplay between light and cold signaling, and in plants, cold temperature has been shown to disrupt or 101 ‘gate’ the circadian clock.Although all plants were grown in the same short day conditions, daylength could amplify an adaptive response induced by BaP. HORMONES Auxin-related response genes were altered in Arabidopsis after 24 h BaP exposure, with a greater proportion up-regulated than down-regulated. The significance of this is not entirely clear, although over half of auxin-responsive genes have been found to be rhythmically expressed, suggesting that auxin response is controlled by the clock.^^^ Given that estimates of circadian regulated genes in Arabidopsis are between 10% and 16%,^^^ this is an extremely large fraction. In the BaP response, more of the auxin- responsive genes that were expressed at higher levels in the controls were circadian regulated (23 out of 38), while those up-regulated by BaP were mostly not circadian (44 out of 46). These data suggested that auxin levels were higher in BaP-exposed plants, which accords with previous findings that BaP concentrations that were not high enough to inhibit growth produced higher auxin levels in ferns.Covington and Harmer (2007) concluded that auxin signaling is regulated by the clock, but the clock is probably not regulated by auxin. The AhR agonist dioxin disrupts the clock in animals,^^® and genes involved in Phases I-III xenobiotic metabolism are regulated by circadian bZip transcription factors.^®** In Arabidopsis, BaP appears both to disrupt the clock and to increase levels of auxin. Therefore, the auxin-related genes that were decreased in 24 h BaP exposure are likely to be down-regulated by the circadian clock, as an indirect effect of BaP. Regulation of the circadian clock is possible by a compound related to auxin metabolism. Recent progress in the decades-old search for an endogenous AhR ligand 102 indicates that the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) has the highest AhR affinity of any compound tested.’®' Exposure to FICZ led to an increase in CYP450 and other transcriptional targets of ligand-activated AhR, and also altered expression of BMAL3 and Per clock genes.’®’ Furthermore, auxin, FICZ, and the circadian regulator melatonin are all tryptophan derivatives. Perturbation of tryptophan metabolism may itself affect circadian rhythms. Circadian cycling of melatonin and its oxidative metabolite AFMK have been found in water hyacinth,’®’ and the authors speculate that high levels of these compounds may contribute to this plant’s exceptional phytoremediation abilities. Growth media supplementation with Trp resulted in higher melatonin synthesis. Melatonin has been found to have auxin-like effects in plants,’®"* and is synthesized at significantly higher levels in cold temperatures.’®® Lei et al. (2004) found that melatonin prevented cold-induced apoptosis in carrot suspension eells, possibly by inereasing levels of polyamines. Auxin also inereases polyamine synthesis, so it is likely that auxin, melatonin, and polyamines act together in some way in cold and circadian signaling.’®® ABA biosynthesis genes were down-regulated by BaP in the microarray data (Appendix D). This did not preclude extensive ABA signaling. The ABA response element, ABRE, was over-represented in 1 kb and 3 kb upstream of genes inereased by BaP response. The most flexible consensus ABRE sequence, [C/A]ACG[C/T]G[T/C/G], may be targeted by HLH, bZIP, and Myc transcription factors in response to multiple hormonal signals. It is difficult to untangle the exact factors involved, but the elear down-regulation of ABA synthesis implied that another hormone signal was involved. 103 Ethylene appears to be an important signal in the short-term response to BaP. The transcript for an enzyme that eatalyzes the first committed step of ethylene synthesis, ACS6, was 2.9x greater in 24 h BaP-exposed plants. The ethylene response elements, ERE, were greatly over-represented in the 3 kb upstream region of genes increased by BaP exposure. Many ERE-binding proteins (EREBP), also known as ethylene response factors (ERF), were up-regulated in BaP response. Ethylene acts as a second messenger or co-hormone with cytokinin, brassinosteroid, auxin, JA, MJ, and SA, and extensive cross-talk exists between ABA and ethylene.’*’ Nevertheless, it seemed that along with many other biotic and abiotic stress responses, ethylene did aet in the BaP response. Intuitively, since ethylene is involved in senescence, abscission, ripening, and selective growth inhibition in the developing hypocotyl, it would be expected to be involved in BaP response. In an unsuccessful response to a carcinogen, an organism would experience uncontrolled growth or proliferation, suppression of apoptosis, and inhibition of differentiation. Plants, more than animals, need plasticity to be able to isolate a foreign body by walling it off, or even abscising an infected organ such as a leaf It is also significant that BaP and ethylene are both combustion products, with cars estimated to produce approximately 90% of airborne ethylene.’** Abeles et al. (1971) measured an increase in beta-l,3-glucanase activity in bean plants exposed to unfiltered air containing elevated levels of ethylene, and also in response to 10 pL/L ethylene for 24 h. Although they described the enzyme as 3.2.1.6, (equivalent to GH16) they only measured activity as glucose released from laminaria.’** A member of glycosyl hydrolase family 17 (GH17), At4g 16260, was up-regulated in 24 h BaP exposure. Members of GH17 (3.2.1.39) can also hydrolyze laminaria. Although our research was separated by almost 104 40 years, we seem to have measured a similar plant response to air pollutants. A beta- glucan oligomer produced from laminaria by a beta-l,3-glucanase had antiproliferative effects against human myeloid leukemia cells. GENES P450 At3g28740 is a member of the CYP450 genes known for their detoxification activity in all phyla. XRE and AML motifs occur in the promoter region of this gene, with no EE motifs. This gene was up-regulated in 24 h BOA treatment, but the authors only identified W-box (WRKY transcription factor binding sites) and G box promoter elements as putative regulatory sites.” ® Elicitation with cis-jasmonate resulted in high expression of this P450, and changed the chemical profile of emitted volatile compounds which repelled herbivorous insects and attracted insect parasitoids.” ' ABC/MRP14 and UGTl Although the ABC transporter, At3g59140, is not induced in 24 h by bleomycin and mitomycin C (eFP Browser), it is induced 1.38x by oxidative stress. The ABC transporter family is well conserved and the closest human homolog to this protein is the multidrug resistance-associated protein MRP2 (e=5 x lO'^-H). MRP2 is induced in response to many different xenobiotics, and associated with resistance to chemotherapy via enhanced drug clearance.” ’ Human MRP2 has been shown to be induced by quercetin but not by dioxin.” ’ It is possible that this gene was not induced directly by BaP, but by a plant flavonoid metabolite downstream of the initial chemical interaction. In the same study, quercetin also induced UGT1A6, a member of the UGTl family 105 capable of glycosylation of quercetin and other compounds. UGT1A6 was induced by dioxin, as UGTl was induced by BaP in our study. Quercetin has antitumor effects” "’ due to antioxidant potential and probably via its induction of MRP2, which helps to clear carcinogens from the body. The AtUGTl promoter contains two AML motifs, one XRE, and an EE. UGTl and other enzymes involved in conjugation of both xenobiotic compounds like BaP and native flavonoids were induced by BaP in 24 h. UGTl localizes to the callose synthase complex during deposition of the phragmoplast.” ® UGTl was identified in a screen for SA-responsive genes, believed to be regulated by bZIP transcription factors in an NPRl- independent process.” ® It was up-regulated in 24 h exposure to the allelochemical benzoxazolin-2(3H)-one (BOA), along with many other putative detoxification genes.” ® It has also been shown to make glucose esters of para-aminobenzoate (pABA), which appeared to be a vacuolar storage form of the folate precursor and sunscreen chemical.” ’ Similarly, UGTl can glucosylate ABA,” * presumably to limit the active pool of the hormone. GH17 At4gl6260 is a member of glycosyl hydrolase family 17 (GH17). A similar gene is up-regulated very strongly in tomato leaves by ethylene, and less by MeJA.” ® Oligosaccharides produced by this enzyme may induce defense response genes, based on evidence from plant pathogen-derived saccharides.’*® GH17 also contains an RGD motif, which could be involved in interaction with the EGF repeats of At 14a in an integrin-like signaling pathway. GH17 was up-regulated at both exposure times. 106 A tU a Atl4a contains an integrin-like domain that may interact with genes containing RGD or KGE motifs. RGD is the motif bound by integrin in animals, which transduces signals from EGFR and other cell surface receptors. This signaling is crucial to invasion and metastasis,^*' with higher levels of integrins produced in tumor tissues. RGD analogues are used to image tumor cells and neovasculature^*^ and are under investigation as therapeutic tools.^*^ RGD, and KGE, which is a variant with similar amino acid characteristics, have been shown to function in cell wall signaling in plants,^*"* and the KGE motif was over-represented in genes up-regulated in 24 h BaP response (18% of genes called increased, vs. 8% in the whole proteome). At4s33980 and At5e42900 Both of these proteins contain KGE motifs, and have unknown functions. At4g33980 (called E4 in Figures 6.1 and 6.2) was highly up-regulated by BaP in 24 h, and contains many potential regulatory motifs in its upstream sequence, including XRE, TATA, EE, AML alternate, G BOX, GBLE, WRKY, and two motifs defined as DNA-damage inducible. 285 It is similar in sequence to At5g42900, (E5 in Figures 6.1 and 6.2), which was also highly up-regulated. It contains the same general battery of motifs, except an AML consensus instead of the alternate motif, and no XRE. Neither belongs to the common stress response gene cluster N 12, delineated by Ma et al. 2007.^*^ (None of the down-regulated genes, and only 26 of 143 genes up-regulated in BaP response, were in this cluster). Instead, they clustered with circadian genes in N52. The core clock gene TOCl is coregulated with them. The other gene of interest in this group is FKFl. 107 FKFl rAtlg68050) The probe set for FKFl also targets two other loei (AT5G23410 and the pseudogene AT5G42730), but since FKFl up-regulation was confirmed by qRT-PCR, only Atlg68050 will be discussed. FKFl is a flavin-binding keleh repeat F box protein that contains two PAS and one PAC domain. E3 ubiquitin ligases help degrade proteins in a clock-dependent marmer. FKFl enables degradation of CONSTANS proteins in the control of flowering time, but its function in BaP response is uncertain. It is a likely candidate for direct binding of BaP through its PAS domain, thereby initiating the disruption of clock timing, which in animals has been shown to correlate with increased cancer risk. 287 Quite recently, the Per2 gene has been defined as a tumor suppressor in breast cancer,’** and shown to target estrogen receptor alpha for degradation. Per2 is also up-regulated by estrogen, and is a major factor in estrogen receptor-positive breast cancer and in normal reproductive health.’*" Where Per2 is a PAS-containing clock protein that interacts with female hormones, FKFl is a PAS-containing protein that affects floral induction and clock signaling.’"" Therefore, FKFl could be the site of BaP interaction with Arabidopsis that perturbs the clock and initiates xenobiotic response signaling. FKFl increases in cold treatment, and to a lesser extent in genotoxic stress (eFP Browser). FKFl could also be a functional AhR homolog, since the AhR was recently found to act as a ligand-activated E3 ubiquitin ligase.’"' TOCl TOCl is part of the central oscillator of the plant circadian clock.’"’ The observed up-regulation of TOCl (Figure 6.2), or perhaps the oscillatory shift in its expression, in response to BaP, accords with recent findings in animals that DNA damaging agents 108 perturb the clock.’”’ Gamma radiation advanced the phase of Perl and Perl expression in rat cells, while MMS treatment disrupted the clock largely by indueing higher Per gene expression. DNA damage is proposed as a universal clock regulator. The mechanisms behind clock dysregulation are not elear, but BaP binding to PAS-eontaining FKFl or another endogenous factor could be involved. G/?F7(AT2G216601 Grp7 belongs to a family of genes, conserved in eukaryotes, that are induced by cold and have important functions in immune response.’”"* Endometrial carcinoma is associated with reduced or no expression of the human HomoloGene for Grp7, CIRPP^ CIRP is up-regulated by UV and the UV-mimetic compound A-acetoxy-2- acetylaminofluorene.’”® CIRP is induced by eold or hypoxia, but does not require activation by HIFl.’”’ In hypoxia, osmotic, or oxidative stress, CIRP migrates to the cytoplasm and aggregates in stress granules.’”* Methylation of arginines in the RGG domain of CIRP is required for translocation of the protein from the nucleus to the cytoplasm, where it binds mRNA and represses translation.’”” The arginine methyltransferase involved is a PRMTl,’”* and the Arabidopsis functional homolog, PRMTl 1,’”” was also up-regulated following 24 h BaP exposure. CIRP homologs were diumally regulated in bullfrog’”' and in mouse brain,’”’ and diurnal rhythms observed in treefrog brain and eye appeared to be gated by eold and light.’”’ Like CIRP, Arabidopsis Grp7 links circadian rhythm with stress response pathways. Grp7 transcription increases in cold and drought.’”"* It is autoregulated by its own protein, whieh increases transcription of a short-lived, alternatively spliced mRNA.’”® The high conservation of this RNA binding protein was demonstrated by studies that 109 showed that both Arabidopsis and human transportin-1 proteins facilitated nuclear import of GRP7, and interacted with human hnRNP Al and yeast Nab2p.’®® In plants, some glycine-rich proteins are localized to the cell wall matrix,’®’ and GRPs have been identified in the xylem sap of different plant species.’®* These proteins are incorporated into the cell wall during xylem formation and cell wall remodeling.’®® GRP7 is highly expressed in trichomes and guard cells, regulating stomatal opening and closing,” ® which places it at the interface with external environmental forces. RD29A The transcription factor RD29A (also known as COR78) is a major regulator of stress response.’” It functions at the intersection of signaling pathways for cold or ABA, and drought or salt responses. Cold or ABA can activate DREBIA. In response to stress (drought, salt), ADRl is up-regulated, activating DREB2A. In Arabidopsis, ADRl was 1.4x up-regulated in the 24 h response, DREB2A was 1.6x up, and DREBIA was 1.9x up. DREBIA and DREB2A are transcription factors that can bind A/GCCGACNN boxes, activating transcription of known stress response genes, and both activate RD29A?^^ Figure 6.3 shows the central role of RD29A in abiotic stress signaling pathways mediated by DREB transcription factors. BoCAR6-4 Atlgl7665 is the Arabidopsis homolog of a gene that was identified in a screen for transcriptional responses to controlled atmosphere treatment (10% C02 + 5% 02; CA).’” CA is a postharvest treatment used to delay the onset of senescence in broccoli and some other commodities. The gene referred to as BoCAR6-4 {Brassica oleracea controlled atmosphere responsive) is up-regulated strongly as an early response to CA, 6 110 to 48 h after treatment onset.*’* Since BaP caused a delayed senescence in the plants grown in agar containing BaP in enclosed jars, a gene that apparently contributed to this phenomenon in a related crucifer had parallels. In broccoli a decrease in expression of 3 other CA responsive genes occurs when exposed to drought, salt, or cytokinin, but B0CAR6-4 was only expressed in CA treatment. In the 24 h Arabidopsis microarray results, Atlgl7665 was called ‘absent’ in all control slides. Since Atlgl7665 contains an ROD tripeptide, it is implicated in cell wall signaling with an integrin-like protein such as At 14a. Hypoxia responsive pathways in animals can be impacted by BaP exposure. For example, BPQ disrupts HIFla and HSP90 association, causing increased proteasomal degradation of HIFla, and leading to decreased KEGF transcription.*’“' Small hydrophobic protein At4g30650 is a small hydrophobic protein with two transmembrane domains. There are two XRE motifs in the upstream region, and an EE. It is reported to respond to salt stress within 4 h,*'* and cold stress within 24 h.*’* It has high BLASTp homology to proteins from bacteria, yeast, and C. elegans, but the function of these proteins appears to be unknown. It is highly coregulated with GRP, and produced one of the highest BaP- responsive transcript levels measured by qRT-PCR. If BaP can bind a PAS-containing protein in Arabidopsis, it could up-regulate this gene via either or both XRE motifs. Although its function is unknown, it is linked with both cold and circadian response pathways through Grp7. Vamp? 13 At5glll50 eodes for a vesicle-associated membrane protein, VAMP713, that contains the conserved eukaryotic longin domain. This domain functions in the AP2 111 complex in clathrin-mediated endocytosis.’" The closest human protein by BLASTp comparison is VAMP7/Sybll (62% positives, with an expect value of 5e-40). VAMP7 has recently been implicated in transport of degradative matrix metalloproteinase to metastatic invadopodia, enabling cancer cells to degrade tissue that otherwise would form a barrier to breast cancer invasion."* In Arabidopsis, VAMP713 could be involved in vesicular trafficking of BaP metabolites, flavonoids, or ROS. Knockout or suppression of VAMP713 and two related genes increased salt stress tolerance in Arabidopsis roots, and reduced transport of H2O2 to the tonoplast."® This gene has the most highly correlated expression with GRP7 across all experiments in the ATTED-II database, further implicating both genes in xylem transport.” ® WAKl Atlg21250 codes for wall-associated kinase 1. WAKs have an extracellular region and a cytoplasmic kinase domain,” ’ and are potentially involved in cell wall signaling. WAKl has a calcium-binding EGF domain, which implicates it in possible interactions with proteins containing RGD or KGE peptides. The EGF domain also contains an Asn or Asp beta-hydroxylation site conserved in Notch, EGF, LDL, and other proteins involved in BaP response in animals. Lack of hydroxylation at this site is associated with cancer.” ’ WAKl has been shown to be essential for pathogen or SA response,” ’ and for cell expansion.” "* Glycine-rich proteins interact with WAKs.” ® In vitro binding assays demonstrated that WAKl was bound by GRP3, but not by GRP7. The results for GRP7 are not conclusive, as only in vitro translated products were tested and the GRP7 product contained at least 7 bands, the largest of which exceeded the predicted size.” ® 112 Jmj /At3g20810 Jumonji C (JmjC) proteins can hydroxylate the crucial B-Asn or Asp sites within EGF domains of proteins like WAKl. Factor inhibiting HIFla (FIH) is a JmjC-containing Asp- or Asn hydroxylase that represses binding of HIF1-coactivating CBP/P300 in normoxia; absence of the hydroxylation leads to HIF la-dependent hypoxia signaling. At3g20810 is the HomoloGene for JMJD5, which has been suggested to function as a tumor suppressor in humans.Conversely, other human proteins containing JmjC domains are posited to be oncogenes. Tsuneoka et al. 2002 showed that c-Myc binds to an E box in the promoter region of MINA53 in proliferating promyelocytic leukemia cells, and that knockdown of MINA53 decreased proliferation. The gene was highly expressed in coal miners and lung cancer cell lines, and knockdown delayed G1 to S phase transition.*^* More recently, JmjC proteins have been identified as histone demethylases. Pfau et al. (2008) concluded that JmjC-containing histone demethylases may perform both tumor suppressor and oncogene functions.**® In their study, a different JmjC protein suppressed senescence via histone demethylation, indirectly promoting phosphorylation of the retinoblastoma protein (Rb) that promotes the G1 to S transition. Histone demethylation is likely to have far-reaching epigenetic consequences, and is a promising field in cancer research. BT4 A second gene that has the potential to cause broad epigenetic changes is BT4. At5g67480 is one of five BTB and TAZ domain containing proteins in Arabidopsis. Although each of these domains is well conserved among eukaryotes, homologous proteins with both domains are only known in plants. TAZ (Transcription adaptor 113 putative zinc finger) domains are found in human P300 and CREBBP transcriptional coactivators, which acetylate histones at lysine residues, activating transcription by providing binding sites for bromodomain-containing transcription factors. These coactivators also respond to signals by acetylating DNA-bound transcription factors, including P53 and E2F. P300 has been reported to ubiquitinate P53 and lead to its degradation, but also has tumor suppressor activity.” ” P300/CREBBP interacts with ARNT in the AhR/ARNT complex.^^' Data suggest that BTB-containing proteins in Arabidopsis may constitute part of the E3 ubiquitin ligase complex. Researchers found that the BTB-TAZ proteins respond to SA and H2O2 , bind calmodulin (CaM), and interact with BET proteins, which are conserved bromodomain-containing transcriptional activators. HSP70 The HomoloGene for human HSPA2 and murine HSP70-2 was down-regulated by BaP in 24 h microarray results. HSP70 genes have been investigated for many years as potential biomarkers for cancer. Elevated levels are found in many cancer types, and siRNA knockdown of HSP70A2 resulted in a senescent phenotype and cell cycle arrest at G2.^” A different group found that BaP led to a dose-dependent decrease in HSP70A2 expression in endothelial cells from pig aorta, and hypothesized that it might occur via protein kinase C signaling and a down-regulation of HSFl.^^® Summary The initial interaction of BaP with Arabidopsis could involve transport by the ABC transporter MRP 14, or vesicle-associated membrane transporter VAMP713. BaP might also bind an unidentified AhR functional homolog such as the PAS domain-containing 114 FKFl, that could direct the transcription of the following genes with XRE motifs in their promoters: E4, hp, Jmj, P450 and UGTl. In animals, BaP drives transcription of the genes whose proteins will directly interact with it; CYP4501A1, COX-2. Therefore P450 may oxidize BaP, while the small hydrophobic protein hp may physically associate with BaP, perhaps helping to sequester unmodified regions of the chemical. GRP7 and hp are coregulated, so may be involved in a process that utilizes their hydrophobic domains. If hp is induced by BaP binding to its XRE motifs, the hp-GRP7 association could be the point of induction of the cold response signaling, and even of perturbation of the clock. This would resemble the mammalian model, where binding of small hydrophobic ligands to nuclear hormone receptors couples circadian rhythm with metabolic functions.*** FKFl is an E3 ubiquitin ligase that interacts with the core of the circadian clock, and might dysregulate or degrade TOCL JMJ may demethylate histones and suppress senescence, as some animal JmjC proteins do,*’" or it may suppress proliferation.**’ Exposure of Arabidopsis and other plants to BaP might alter cell wall signaling and wall remodeling, as BaP can be incorporated into lignin.*** In animals carcinogenesis is associated with a perturbation of EGFR and integrin-mediated signaling leading to cell proliferation. EGF binding is inhibited by BaP through a reduction in EGFR protein,**" and possibly through a reduction of VEGF by BaP-induced proteasomal degradation of HIFla, which would otherwise induce VEGF?^^ In plants, the EGF-containing proteins At 14a and WAKl may direct these processes by associating with RGD and KGE tripeptides in BoCAR (shown to inhibit senescence in broccoli), GH17 (may produce biologically active carbohydrate moieties), and MRP 14 (transporter/ATPase). BT4 may 115 interact directly with BaP as human P300 does via its TAZ domain, resulting in altered histone acetylation and aberrant transcription. The failure of single-gene cancer targets to produce effective antitumor treatments, plus reeent advances in understanding DNA methylation changes and histone modifications has led to growing interest in epigenetie meehanisms of eareinogenesis. Chemotherapeutic histone deacetylase inhibitors are being investigated for their potential to combat various cancers.’*' Microarray data are providing pathway-based targets in addition to SNP or oncogene targets.’*’ Even the field of nutraceuticals is going wide in the search for epigenetic phytochemical effects.’*’ BaP altered transcription of Arabidopsis genes that are homologous to genes known to be involved in animal response to BaP. BaP-indueed stress response pathways overlap with better-characterized abiotic stresses, but differ in ways that have been minimally explored with xenobiotic chemicals. The alteration of auxin signaling pathways may yield information about the function of auxin-like compounds such as melatonin in plants, and their interactions with circadian regulation. The identification of a JmjC- containing gene and a BTB/TAZ gene as potential effectors of BaP response in plants eould provide additional information that helps delineate the epigenetic effects of BaP. 116 CHAPTER 7 Summary and Future Directions 7.1 Concluding Remarks a. Cross-kingdom comparisons For those who are uninspired by the universality of basic processes, for whom the discovery of the homeobox was not a religious conversion, this work may seem far fetched or impractical. After all, why waste time on plant models when there are cell lines, animal models, and epidemiological data to work with? The main line of argument with this position is that sometimes it is necessary to think about other systems, to draw examples from other fields, in order to better understand our own narrow field. A good example comes from molecular biology. In the early days, researchers were excited to work with DNA, the code itself Some of us work with RNA. More recently, proteomic and metabolomic methods have enabled biologists to access products of the genetic code. Ironically, as these methods developed, the older chemical assays for protein activity were neglected. We now had positive identification of the peptides, but had to go back to the ‘old’ activity assays to show their relevance. Similarly, new functions for ‘non coding’ DNA and RNA continue to be proposed and discovered. The reeent prediction that most of the human genome is transcribed,” '* instead of the 1-2% that was previously estimated,’'*® gives new dimension to the concept of the ‘RNA World’, the theory that RNA formed the original building blocks of life.’'*® The basis of biology is chemistry, and there may be an inherent processivity to transcription that ensures massive pools of RNA will be produced. Whether that RNA has a function will depend on biological selection. 117 More recently, a theory of a ‘PAH World’ was proposed, by an astrophysicist.*"” Astrophysics doesn’t mix much with molecular biology, but maybe it ought to. Basically, the theory is built on the fact that PAHs are ubiquitous, even in distant galaxies, beeause of planetary, galactic, and maybe even intergalactic combustion processes (collisions, explosions, all that space stuff). Also, planar PAHs form stacked structures that are oriented 3.4 angstroms apart (like a double helix). These structures form spontaneously, with more hydrophilic or oxidized regions on the outside, in an energy-minimizing fashion. Pyrimidines and purines are predicted to attach to the outside of the PAH stacks, forming a nucleic acid backbone, followed by sugars. If this sounds far-fetched, remember the attraction that BaP has for nucleic acids (especially G and A). A survey of tumor transcriptomes found a majority of mutations (more than 53%) oecurred at C:G to T:A, with most of these (37%) occurring within CpG sites.*"*® There is no reason to assume the molecular attraction between DNA and BaP oceurs in only 1 direction. b. Viruses and BaP A recent study found a novel mechanism for BaP involvement in cervical cancer. While BaP was known to be physically present in cervical mucus of smokers, who develop the cancer at higher rates, for the first time researchers showed that the HPV viral genome replicated faster in the presence of low concentrations of BaP.*"** Increased genome replication of a DNA virus could indicate epigenetic mechanisms at work. It is possible that BaP is physieally interacting with the DNA to enhanee replication. The phenomenon eould involve molecular affinity in a cooperativity or processivity-like 118 effect. Given the co-involvement of vimses and carcinogens in cancer, a direct interaction between the two would not be surprising. c. Benefits of BaP? Vimses can be thought of as evolved nucleic acids. If DNA or RNA can evolve to benefit from the chemical and physical properties of BaP, can eukaryotic organisms also do so? The finding that BaP could extend the lifespan of Arabidopsis was novel. Animals do not generally benefit from BaP exposure, but there is one report of BaP having a protective effect against endometriosis in uterine tissue, by decreasing levels of cell adhesion proteins and epidermal growth factor receptor (McGarry et al., 2002).’®® In an immune response, BaP has an adjuvant effect on antibody production, but the mechanisms for this are not yet understood.’®’ Possibly, the plants grown in agar with 50 ppm BaP lived longer than the controls because of a similar immune-stimulating effect. These plants did demonstrate up-regulation of defense response genes along with immune-enhancing down-regulation of NudtV. d. Plant-specific responses Cytochrome P450 enzymes convert BaP to the mutagenic diol epoxide form in animals, and possibly in plants, but what makes the difference in survival between plants and animals? When pre-neoplastic mammary cells were treated with tea epigallocatechin gallate (EGCG), the soy isoflavone genistein, or the cmciferous glucosinolate indole-3- carbinol plant compounds, the aberrant proliferation induced by 24-h BaP exposure was inhibited 44 to 65%.’®’ Quercetin inhibited BaP-induced DNA adducts by repressing CYPlAl mRNA and protein expression.’®’ Production of flavonoids might explain why so few of the 246 CYP450s in the Arabidopsis genome are up-regulated.’®"* The 119 GeneChip expression profiles indicated that glucosinolate production was up-regulated in 24-h BaP response and flavonoid pathways were increased at 4 wks, especially in the profile of one of the three BaP-exposed biological replicates. This indicates that chemoprotective compounds may act within the plant in ways similar to their activities in animal systems. The anticancer alkaloids vincristine and vinblastine are produced by a pathway that includes strictosidine. The up-regulation of strictosidine synthases after 4 wk exposure to BaP suggests that plants may have evolved chemical defenses against environmental carcinogens, even if Arabidopsis does not retain all functional members of the pathway Some genes that responded to BaP are unique to plants, including germin-like proteins, lipid transfer proteins, osmotins, HINl, Clavata 3-like CLE12, arabinogalactans, expansins, and ethylene-, ABA-, and auxin-responsive proteins. Generally, few of these genes have been shown to respond to PAH, simply because they have not been measured. Alkio et al. 2005 measured decreased EXPA8 expression in Arabidopsis grown in phenanthrene. This gene was down-regulated in 24 hour BaP exposure, as was EXPAl, but neither was consistently changed in 4-wk exposure. These two genes are often co regulated, according to the ATTED2 database, and in fact show close correlation (nearly identical expression values) between individual 24-h replicate samples. Alkio et al. also measured an increase in PRl expression, indicating SA activity, and correlating with their measurement of cell death and hypersensitive response. The symptoms they described (white spots, necrosis) were not observed in any of the BaP-exposed plants, and it seems from the expression profiles that ethylene, JA, and ABA probably play bigger roles in BaP response than SA, at the timepoints tested. 120 e. Microarrays identify cancer pathways The microarray analyses identified candidate genes and pathways present in the plant genome. The candidate genes were involved in uptake, transport, sequestration, or biochemical conversion of BaP. Identification of pathway elements may provide the basis for developing a parallel model for studies of chemical carcinogenesis or for toxicological measurements that preclude the use of animals. Recent epidemiological microarray-based analyses of tumor transcriptomes have shown the importance of pathway regulation in cancer development.^®® For a field which has typically targeted individual tumor suppressors or oncogenes, this was a new paradigm. Although PI3K itself is a tumor suppressor, the entire PI3K/MTOR/rps6- kinase pathway was shown to be highly up-regulated in aggressive breast cancer.®®® Analysis of pancreatic tumors revealed an average of 63 mutations per tumor, which is discouraging for therapies that seek to target a single mutation.®'** The bulk of the mutations occurred in genes within 12 common pathways, with each pathway altered in 2/3 to all of the tumors. Many of the pathways have already been implicated in carcinogenesis, but the research confirmed that adhesion and integrin-related pathways were dysregulated in most of the tumors. The results of the Arabidopsis arrays indicate dysregulation of RGD and EGF-containing proteins that could be involved in similar pathways in plants. Manipulation of these pathways in Arabidopsis could help assess effects of potential cancer treatments on other signaling pathways, since many pathways are common to plants and mammals. Therapies that target a pathway rather than a single mutated gene, and that use agents with broad transcriptome effects like histone deacetylase inhibitors appear promising.®®^ 121 Gene-targeting approaches to cancer therapy are also threatened by the microarray- enabled discovery that tumors evolve. 358 The initiating or promoting mutations may be lost as the tumor grows, meaning the tissue will be heterogeneous for that gene, and potentially for entire pathways. Despite these new methodological complications, microarrays provide one of the best means to visualize what’s going on. 7.2 Obstacles and future directions a. Comet assay The alkaline comet assay was able to detect differences in DNA migration from lysed nuclei of control and BaP-exposed plants. The assay is inherently non-specific, and risks the possibility of measuring artifactual differences such as ROS-induced lesions. Most of the more gene-specific methods depend on the availability of expensive equipment like sequencers and mass spectrometers, which are capable of detecting mutations at particular loci. Less expensive methods are rare, but include a version of comet assay. Comet assay coupled with fluorescence in situ hybridation (comet-FISH) is a powerful tool to determine if specific genes are mutated. Mutations in P53 were detected in human colon cells by comet-FISH, using fluorescent probes for P53. The technique allowed visualization of preferential DNA cleavage at P53 (vs. global DNA damage) when treated with mutagens, and also showed DNA repair when the cells were harvested further from the initial exposure time (probes hybridized to comet heads, not tails).**" Although there is no identified plant P53 homolog, there is some evidence for the existence of a functional homolog.**" A putative tumor suppressor-like gene that showed low or variable expression levels in microarray analysis might be worth 122 investigating with this technique. For example, At2g02710, which codes for three variant PAS/LOV domain proteins, was decreased in 4 wk BaP shoot tissue, to different degrees. The tumor suppressor Per2 has a PAS domain, so it is possible that this domain could have a similar function in At2g02710.’®' NudtS is the HomoloGene for human Nudt6/GFG, which suppresses proliferation. It was down-regulated at 4 wks in two of three BaP-exposed samples, and might also be worth further study. The best candidate would probably be TAZ/BT4, which was up-regulated at 24 h, and in one of the 4 wk samples grown in BaP, but decreased in the other two. BT4 may act similarly to mammalian P300, which also contains a TAZ domain, responds to BaP, and has tumor suppressor activity.’®’ b. SAandNSAIDs Additional work could include investigation of the effects of aspirin or other non steroidal anti-inflammatory drugs (NSAIDs) on the plant-BaP interaction. Specifically, it is hypothesized that the alpha-dioxygenase DOXl or its homolog, Atlg73680, may be repressed by NSAIDs that repress the homologous COX-1 and COX-2 genes in humans to different degrees, and have anticancer activity.’®’ The two Arabidopsis genes show different induction, as indicated by experiments archived in the eFP Browser. Atlg73680 is increased 1.5-fold by ibuprofen, but decreased slightly by SA, in 3 hours. DOXl is increased 2.7-fold by SA, but showed no response to ibuprofen in 3 h. Since this is an ongoing pharmaeeutieal and medical concern, any information gained from the plant model will be of interest. Recent work with analogs of the selective COX-2 inhibitor celeeoxib has shown that at least part of its antieaneer activity comes from the non-COA- 2 inhibitory moiety, and these analogs are being developed as cancer treatments.’®* The 123 mode of action of these analogs appears to be induction of ER stress and restoration of contact inhibition,’®® which may involve RGD and EGF signaling. c. Protein assays Many of the genes whose expression was altered by BaP would be worth further study. The CYP450, peroxidase, DOXl, and laccase genes could be assayed for enzyme activity against BaP. Protein binding or affinity to BaP could be tested using Biacore fixed platform or KinExA fluidics systems. Candidates for these types of assays include ABC, MRP3, LTP, and AGP24 with BaP or metabolites. It would also be informative to test binding of Atl4a or WAKl with the RGD and KGE peptides within GH17, E4 or E5 proteins. The AtNap KGE domain may show similar affinity to the C2 domain of PLD2 as cardosin A from cardoon.’®® For FKFl, many aspects of the protein should be checked: affinity for BaP, and interacting partners for clock and E3 ubiquitin ligase functions. A detailed time course experiment using at least FKFl, TOCl, and potential binding partners would be helpful. Gel shift assays could be used to check if there is any binding to the canonical XRE. d. Proteomic profiling Proteomic profiling has the potential to identify more metabolic effects of BaP, since it detects actual protein levels. One drawback of transcriptional profiling is that it cannot determine enzyme activity of the gene product, or account for post-translational modifications or proteolytic degradation. For example, higher COX-2 transcript levels are irrelevant if the protein is not produced, phosphorylated, or has no activity. Protein profiles would give information about protein abundance and modifications, and 124 detection of protein multimerization is possible. Additional assays are needed to determine if it associates with other molecules or is simply degraded, e. Detecting transcripts after treatment with a mutagen The core difficulty of this project was that BaP could cause mutations that interfered with transcription or prevented it altogether. Variable mutation occurrence in a study with a low n could be expected to produce erratic microarray signals. The mutation rate in Arabidopsis, especially via the administration route used here, was unknown. By using standard statistical approaches to discard genes with irregular expression across the sample set, one risks discarding the very genes that matter; those that are mutated in direct response to the carcinogen, and whose mutation mediates downstream effects. Many of the most interesting gene candidates, i.e. those potentially involved in mutagen response, were not consistently detected in the GeneChip assays. For example, homologs of tumor suppressor genes {Rb, BRCA, MOBKLIA, Wilms’ tumor suppressor, FHIT, 0VCA2) were either called absent or had varying patterns of up- and down- regulation within a single subset of BaP-exposed samples. Most commonly, a single BaP-exposed sample would exhibit down-regulation. The retinoblastoma homolog AT3G12280 was decreased at 4 wks in one replicate. Inconsistent patterns could reflect a low rate of mutations, translocations, or other mutagenic events leading to dysregulation. By looking only for eonsistent, reproducible expression ratios one risks overlooking significant mutation events. With a very high number of replicates, it might be possible to separate genes that appear to be mutated in rare cases, but that seems inefficient. Also, the high correlation of qRT-PCR data with the GeneChip results suggests more replication would be largely redundant. Future studies that include 125 analyses of DNA adducts, methylation, deletions, and translocations could yield a more complete picture of chemical effects and plant responses. Detection of mutations within these homologous genes would be interesting from the standpoint of carcinogen research, as plant models are free of certain ethical constraints related to laboratory use and manipulation of mammalian subjects, while providing a whole-organism context, f. Phytoremediation and cancer As the petroleum-based economy declines,’®’ pollution by PAHs will persist. Although green technologies are moving into the mainstream, it remains to be seen whether bioengineered phytoremediators would be palatable to a public overwhelmed by corporatocracy and alienated from food production. At a minimum, this research should aid in screening and selection of prospective plant species for PAH remediation. The data demonstrate that a small crucifer can tolerate high levels of BaP without negative effects, and that glucosinolate, flavonoid, and alkaloid synthesis pathways were stimulated. This suggests that larger cmcifers may be good remediators, although it should be considered that they may also sequester BaP. It would be interesting to test Rauwolfia serpentina and Catharanthus roseus to see if they are good remediators, and also if PAH induces higher production of reserpine, vincristine or vinblastine. This would be only to probe the pathway, as carcinogen-elicited chemotherapy would doubtless be even less popular than GMO phytoremediation. However, Arabidopsis itself may be a promising bioindicator, since strictosidine synthase Atlg74010 shows strong induction by BaP and is also reported to increase in response to 3-h cycloheximide treatment.’®* 126 Building on earlier evidence of BaP uptake and degradation by plants, this study has measured up-regulation of multiple genes proposed to be involved in uptake and detoxification. Among these are the predicted CYP450, peroxidase, glutathione dehydrogenase, ABC transporters, UGTl, and even a COX-2 homolog. However, the study also identified specific responders from categories as diverse as cold and circadian response (Grpl), jumonji C proteins, glycosyl hydrolase family 17, controlled air responsive, ubiquitin ligase {FKFl), cell wall signaling {WAKl), napsin, Nudix hydrolase, vesicle-associated membrane protein, strictosidine synthase, EGF repeat and RGD containing, and two proteins of unknown function. The qRT-PCR results confirmed that BaP exposure led to a perturbation of the plant circadian clock {FKFl, TOCl, GrpT). Disruption of the mammalian clock in cancer has recently been acknowledged, after years of epidemiological evidence that shift work predisposes to cancer.*^®’*™ Grp7 up-regulation reveals a linkage between a carcinogen signal and a stress response profile associated largely with cold stress, which also impinges on rhythm. The PAS domain of FKFl is a candidate for direct binding with BaP to initiate or perpetuate the circadian disruption. 127 Appendix A. Mammalian genes (or proteins) primarily up-regulated in response to BaP or BPDE Gene Time of Function Binds to/ Biological System Ref. Expression Interacts with aP IX a-PAK PAK; GEF for HeLa cells 15 interacting Cdc42/Racl exchange factor ATF3 TF; homodimers ATF2, c-fos, c- Normal mammary 13 repress, many jun, junB, junD, epithelial cells heterodimers GADDI53, p53 Bax 24 h P53-dependent Lung cancer cell 8 lines BRD3 Putative Serine- amnion epithelial 4 Bromodomain- threonine kinase cells containing 3 Calcium 1 h BaP; 1 Vascular smooth 6 modulating wk recovery muscle cells ligand CD-95 24 h Porcine bladder 14 epithelial cells CdcIA 1 h BaP; 1 Cell division Vascular smooth 6 wk recovery cycle homolog A muscle cells Cdc25B 24 h G2/M transition; Binds steroid Bronchial & lung 9 CDK receptors, EGFR, cancer lines phosphorylation ASK-1 Cdc42 1-6 h Activates JNK-1 HeLa cells 15 Cdc7l homolog 1 h BaP; 1 Cell division Vascular smooth 6 wk recovery cycle muscle cells c-Fos 6 h Liver slices 10 Chondroitin 1 h BaP; 1 Vascular smooth 6 sulfate wk recovery muscle cells proteoglycan CLICl Voltage-gated Anmion epithelial 4 Protein chloride channel cells Clusterin 24 h Apoptosis, TK6 lympho- 1 immune blastoid cells response COX-2 0-20 h Can co-oxidize Porcine bladder 14 BaP epithelial cells C SF-la 24 h Growth factor, TK6 lympho- 1 cytokine blastoid cells Cyclin G 24 h Cell cycle TK6 lympho- 1 blastoid cells C YP IA l 0-20 h; most BaP activation Porcine bladder 14 induced at epithelial cells exposure CYPIBI BaP activation Lung cells 7 Death-associated 1 h BaP; 1 Vascular smooth 6 kinase 2 wk recovery muscle cells DNase inhibited 1 h BaP; 1 Vascular smooth 6 wk recovery muscle cells 128 Appendix A (Continued) DNA-Poip 10&24h Normal mammary 13 epithelial cells ERKl 1-24 h Extracellular RAW 264.7 3 Protein signal regulated macrophage cells MAP kinase; apoptotic control ERK2 1-24 h Extracellular RAW 264.7 3 Protein signal regulated macrophage cells MAP kinase; apoptotic control Folate binding 1 h BaP; 1 Vascular smooth 6 wk muscle cells recovery GADD45 4 & lO h Cell cycle P53-dependent, TK6 lympho 1, 13 but down effector (G2/M cdc2/cyclin BI blastoid cells; at 24 block) kinase inhibition, normal mammary h (1 3 ); up MTK1/MEKK4 epithelial cells at 24 h (l) Gamma-glutamyl 24 h Metabolism TK6 lympho 1 transpeptidase blastoid cells GPX 4&24h Glutathione TK6 1 peroxidase. lymphoblastoid Phase II cells Glutathione 24 h Cofactor/vitamin TK6 1 synthetase /amino acid lymphoblastoid metabolism cells GST, Glutathione 24 h Phase II Regulated by M. musculus and 11 S-transferase detoxification Nrf-2 nrf-2 k.o. mice Growth arrest 1 h BaP; 1 Vascular smooth 6 wk muscle cells recovery Growth factor 1 h BaP; 1 Vascular smooth 6 receptor bound wk muscle cells protein recovery HGP 1 h BaP; 1 Vascular smooth 6 wk muscle cells recovery H-Ras 24 h Proto-oncogene Has electrophile Smooth muscle 2 response element cells, human and (EpRE) & XRE mouse Histocompatibility 1 h BaP; 1 Vascular smooth 6 2 complement wk muscle cells component factor recovery Histocompatibility 1 h BaP; 1 BaP 7,8-diol Vascular smooth 6 2 K region wk induced -40% muscle cells recovery vs. BaP Histocompatibility 1 h BaP; 1 BaP 7,8-diol Vascular smooth 6 L region wk induced -25% muscle cells recovery vs. BaP Histocompatibility 1 h BaP; 1 BaP 7,8-diol Vascular smooth 6 Q region wk induced -30% muscle cells recovery vs. BaP 129 Appendix A (Continued) HMG-I(Y) 1 h BaP; 1 Vascular smooth 6 wk muscle cells recovery Interferon 1 h BaP; 1 Vascular smooth 6 activated gene 20 wk muscle cells recovery Interferon 1 h BaP; 1 Vascular smooth 6 inducible wk muscle cells recovery Interferon 1 h BaP; 1 Vascular smooth 6 regulatory factor wk muscle cells recovery JN K l 15-150 c-Jun NH2- HeLa cells 15 min. terminal kinase 1 JUNE 24 h TF Normal mammary 13 epithelial cells Leukemia 24 h Intracellular TK6 1 inhibitory factor transducer, lymphoblastoid effector, cells modulator Lymphocyte IhBaP BaP 7,8-diol & Vascular smooth 6 antigen 6 complex, exposure; 3,6-BPQ showed muscle cells locus C 1 wk -40% & -20% recovery induction vs BaP Mdm-2 24 h P53-dep. Lung cancer cell 8 lines MDR-1 24 h ABC multidrug TK6 1 resistance lymphoblastoid transporter cells Metallothionein 2 24 h Xenobiotic TK6 1 metabolism, lymphoblastoid stress response cells Microsomal 24 h Regulated by M. musculus and 11 epoxide hydrolase Nrf-2 nrf-2 k.o. mice Myxovirus 1 h BaP; 1 Vascular smooth 6 resistance wk muscle cells recovery NAD(P)H:quinone 24 h Phase 2 Regulated by M. musculus and 11 oxidoreductase detoxification Nrf-2 nrf-2 k.o. mice N qol 1 h BaP; 1 NAD(P)H Vascular smooth 6 wk dehydrogenase muscle cells recovery Nrf-2 24 h TF Binds ARE to M. musculus and 11 induce GST, nrf-2 deficient NQO; mice suppressed by Keap-1 Nuclear protein 9 1 h BaP; 1 Vascular smooth wk muscle cells 6 recovery Oncoprotein 1 h BaP; 1 Vascular smooth 6 induced transcript wk muscle cells recovery 130 Appendix A (Continued) Opioid receptor, 1 h BaP; 1 Vascular smooth 6 sigma wk muscle cells recovery 0X 40 Ligand 24 h Growth factor, TK6 lympho- 1 cytokine blastoid cells P21 24 h; No increase in Lung cancer cell 5 ,8 24 h p21 protein (24); lines(24); breast Increased protein cancer lines(16) (16) P21-W AF Late (24 Gl/S block P53 Normal mammary 13 h) epithelial cells P53 4, 8 & 24 Breast cancer cells 5 h; 24 h P53R2 Late (24 Normal mammary 13 h) epithelial cells P A K l 15-150 P21-activated HeLa cells 15 min kinase PDGF-R-alpha 1 h BaP; 1 Vascular smooth 6 wk muscle cells recovery Pericentrin 2 1 h BaP; 1 Vascular smooth 6 wk muscle cells recovery Phenol 24 h Xenobiotic TK6 lympho- 1 sulfotransferase metabolism blastoid cells R acl 1-6 h HeLa cells 15 RNA Pol I 2h-14 transcription Rat liver 12 days Secreted 1 h BaP; 1 BaP elicited ~11 Vascular smooth 6 phosphoprotein 1 wk fold increase; muscle cells recovery quinone and diol very little SE K l HeLa cells 15 SSAT 24 h Amino acid TK6 lympho- 1 metabolism blastoid cells STAT-3 24 h TF, intracellular TK6 1 transducer, lymphoblastoid effector, cells modulator Sulfotransferase, 1 h BaP; 1 Vascular smooth 6 hydroxysteroid wk muscle cells preferring recovery Thymidine kinase 24 h Nucleotide TK6 lympho 1 metabolism blastoid cells Thymidylate kinase 1 h BaP; 1 Vascular smooth 6 homolog wk muscle cells recovery Tissue factor 24 h Cell surface TK6 lympho 1 antigen blastoid cells Tnf-a 1-24 h apoptosis BaP-i-carbon RAW 264.7 3 Protein black, but neither macrophage cells alone, induced TNF-a (protein) 131 Appendix A (Continued) Transcription 1 h BaP; 1 Vascular smooth 6 termination factor wk muscle cells recovery UGT 0-20 h Phase II Porcine bladder 14 conjugation epithelial cells WIPl 4 & lO h P53 P53-dep; Normal mammary 13 phosphorylation feedback epithelial cells control inhibition of p38 MAPK p53 phosphorylation XPC Early Recognition of Normal mammary 13 (4h) DNA damage; epithelial cells nucleotide excision repair Zfp-14 1 h BaP; 1 Vascular smooth 6 wk muscle cells recovery Zfp-35 Zinc finger TF Amnion epithelial 4 Protein homology cells ZNF184 Zinc finger TF Amnion epithelial 4 Protein cells ZNF189 Zinc finger TF Amnion epithelial 4 Protein homology cells 132 Appendix B Mammalian genes (or proteins) primarily down-regulated in response to BPDE or BaP Tim e of Biological Gene Expression Function Interacts with System Ref. A4 1 h BaP; 1 Amyloid beta Vascular 6 wk recovery precursor smooth muscle cells AR 24, 48, 72 h Androgen AhR/BPDE, Akt may Lung cells 7 receptor ubiquitinate A R P l 10&24h TF Normal 13 mammary epithelial cells BAXa 24 h Repressed by BaP, not Breast cancer 5 BPDE cells Bcl-2 24 h Repressed by BaP, not Breast cancer 5 BPDE cells BRCA-1 24 h Repressed by BaP & Breast cancer 5 BPDE cells C184L-22 mr 1 h BaP; 1 Vascular 6 wk recovery smooth muscle cells Claudin-7 1 h BaP; 1 Vascular 6 wk recovery smooth muscle cells c-Jun 0.5 h Liver slices 10 c-Myb 4h Oncogene, TK6 lympho- 1 transcriptional blastoid cells activator c-Myc 4&24h Oncogene, TK6 lympho- 1 transcriptional blastoid cells activator Complement 1 h BaP; 1 Vascular 6 factor h wk recovery smooth precursor muscle cells Connexin-32 4h Cell-cell adhesion TK6 lympho- 1 receptor blastoid cells Cyclin A 24 h Cell cycle TK6 lympho- 1 blastoid cells Decay 1 h BaP; 1 Vascular 6 accelerating wk recovery smooth factor muscle cells Deoxycytidine 4 h Nucleotide & TK6 lympho- 1 kinase xenobiotic blastoid cells metabolism D-interacting 1 h BaP; 1 Vascular 6 myb-like wk recovery smooth muscle cells DNA 4 h DNA synthesis, TK6 lympho- 1 Topoisomerase 11 recombination, blastoid cells repair 133 Appendix B (Continued) E2A 4& lOh TF P21-Wafl Normal 13 mammary epithelial cells Fern la 1 h BaP; 1 wk Vascular 6 recovery smooth muscle cells Gl ucosylceram ide 4h Lipid metabolism TK6 lympho- 1 synthase blastoid cells Growth Arrested Specific 4h Cell cycle control TK6 lympho- 1 protein 1 blastoid cells GTP binding protein- 1 h BaP; 1 wk Vascular 6 associated recovery smooth muscle cells Heme oxygenase-1 24 h Xenobiotic TK6 lympho- 1 transport blastoid cells Histidine decarboxylase 4h Nucleotide TK6 lympho- 1 metabolism blastoid cells 3-hydroxy-3- 1 h BaP; 1 wk Vascular 6 methylglutaryl-CoA recovery smooth ligase muscle cells HMG CoA Reductase 4&24h Lipid metabolism TK6 lympho- 1 blastoid cells IkB-a 4 h TF TK6 lympho- 1 blastoid cells IAP-1 4h Apoptosis TK6 lympho- 1 blastoid cells Immediate early response 1 h BaP; 1 wk Vascular 6 recovery smooth muscle cells ILGF 1 h BaP; 1 wk Insulin-like growth Vascular 6 recovery factor smooth muscle cells ILG F binding protein 1 h BaP; 1 wk Vascular 6 recovery smooth muscle cells Mannosidase 2aB 1 h BaP; 1 wk Vascular 6 recovery smooth muscle cells Nuclear receptor binding 1 h BaP; 1 wk Vascular 6 factor recovery smooth muscle cells Ornithine decarboxylase 24 h Amino acid TK6 lympho- 1 metabolism blastoid cells PSScdc 24 h Cell cycle, TK6 lympho- 1 intracellular blastoid cells transducer/effector /modulator PEP-ck PEP carboxy Down regulated by 1 kinase; primary ATF3? TK6 cell lines metabolism Prolactin 24 h Hormone TK6 lympho- 1 blastoid cells 134 Appendix B (Continued) Ref-1 24 h DNA damage TK6 lympho- signaling & repair blastoid cells RNA Pol II 2 h-14 days Transcription Compensated by Pol I Rat liver 12 R N A P olIII 2 h-14 days Transcription Compensated by Pol I Rat liver 12 SAMdC 24 h Amino acid TK6 lympho- metabolism blastoid cells Sarcoplasmic 4 h ATPase TK6 lympho- reticulum Ca2+ transporter blastoid cells ATPase SHB/Src 4h Adaptor and TK6 lympho- homology 2 receptor- blastoid eells associated protein Small proline- 1 h BaP; 1 Vascular rich protein wk recovery smooth muscle cells SNF2L1 SWI/SNF related, Amnion Protein matrix associated, epithelial actin dependent cells regulator of chromatin SOX4 24 h TF Normal 13 mammary epithelial cells Transthyretin 4 h Extracellular TK6 lympho- transporter/carrier blastoid cells Ubiquitin- 1 h BaP; 1 Protein Vascular conjugating wk recovery degradation smooth enzyme muscle cells ZNF141 Putative Amnion Protein transeriptional epithelial repressor cells ZNF255 Putative zinc Amnion Protein finger TF epithelial cells References for Appendices A and B 1. 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Jeffy, BD, Chimomas, RB, Chen, EJ, Gudas, JM, and Romagnolo, DF, 2002. Activation of the aromatic hydrocarbon receptor pathway is not suffieient for transcriptional repression of BRCA-1: requirements for metabolism of benzo[a]pyrene to 7r,8t-dihydroxy-9t, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Cancer Res 62:113-121. 6. Jeffy, BD, Chimomas, RB, Chen, EJ, Gudas, JM, and Romagnolo, DF, 2002. Activation of the aromatic hydrocarbon receptor pathway is not sufficient for transcriptional repression of BRCA-1: requirements for metabolism of benzo[a]pyrene to 7r,8t-dihydroxy-9t, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Cancer Res 62:113-121. 7. Lin, P, Chang, JT, Ko, JL, Liao, SH, and Lo, WS, 2004. Reduction of androgen receptor expression by benzo[alpha]pyrene and 7,8-dihydro-9,10-epoxy-7,8,9,10- tetrahydrobenzo[alpha]pyrene in human lung cells. Biochem Pharmacol 67:1523- 1530. 8. Nakanishi, Y, Pei, XH, Takayama, K, Bai, F, Izumi, M, Kimotsuki, K, Inoue, K, Minami, T, Wataya, H, and Hara, N, 2000. Polycyclic aromatic hydrocarbon carcinogens increase ubiquitination of p21 protein after the stabilization of p53 and the expression of p21. JRespir Cell Mol Biol 22:747-754. 9. Oguri, T, Singh, SV, Nemoto, K, and Lazo, JS, 2003. The carcinogen (7R,8S)- dihydroxy-(9S, 10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene induees Cdc25B expression in human bronchial and lung cancer cells. Cancer Res 63:771-775. 10. Parrish, AR, Fisher, R, Bral, CM, Burghardt, RC, Gandolfi, AJ, Brendel, K, and Ramos, KS, 1998. Benzo(a)pyrene-induced alterations in growth-related gene expression and signaling in precision-cut adult rat liver and kidney sliees. Toxicol Appl Pharmacol 152:302-308. 11. Ramos-Gomez, M, Kwak, MK, Dolan, PM, Itoh, K, Yamamoto, M, Talalay, P, and Kensler, TW, 2001. Sensitivity to earcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc Natl Acad Sci USA 98:3410-3415. 12. Shah, G.M. and R.K. Bhattacharya, Modulation of transcription in rat liver by benzo[a]pyrene. Cancer Lett, 1987. 35(2): p. 191-8. 13. Wang, A, Gu, J, Judson-Kremer, K, Powell, KL, Mistry, H, Simhambhatla, P, Aldaz, CM, Gaddis, S, and MacLeod, MC, 2003. Response of human mammary epithelial eells to DNA damage indueed by BPDE: involvement of novel regulatory pathways. Carcinogenesis 24:225-234. 14. Wolf, A, Kutz, A, Plottner, S, Behm, C, Bolt, HM, Follmann, W, and Kuhlmann, J, 2005. The effect of benzo(a)pyrene on porcine urinary bladder epithelial cells analyzed for the expression of selected genes and cellular toxicological endpoints. Toxicology 207:255-269. 15. Yoshii, S, Tanaka, M, Otsuki, Y, Fujiyama, T, Kataoka, H, Arai, H, Hanai, H, and Sugimura, H, 2001. Involvement of Alpha-PAK-lnteracting Exchange Factor in the 136 PAKl-c-Jun NH2-Terminal Kinase 1 Activation and Apoptosis Induced by Benzo[a]pyrene. Mol Cell Biol 21:6796-6807. 137 Appendix C Gene loci identified by microarray as up- or down-regulated by BaP in 4 weeks Locus ID Annotation FC AT3G59930; [AT3G59930, defensin-like (DEFL)]; AT5G33355 [AT5G33355, DEFL] 6.1 AT5G38940; [AT5G38940, manganese/metal ion binding, nutrient reservoir]; 5.9 AT5G38930 [AT5G38930, germin-like protein, putative] AT4G11650 ATOSM34 (OSMOTIN 34);thaumatin domain 4.9 AT1G74010 strictosidine synthase 4.9 A T IG 18980 germin-like protein, putative 4.6 AT4G19810 glycosyl hydrolase family 18 protein 3.6 AT1G21310 ATEXT3 (EXTENSIN 3); structural constituent of cell wall 3.5 AT3G01420 ALPHA-D0X1 (ALPHA-DIOXYGENASE 1) 3.4 AT1G73260 trypsin and protease inhibitor /Kunitz family 3.4 AT4G04460 aspartyl protease 3.3 AT1G65690 harpin-induced protein-related / HINI-related 3.2 AT4G29640 cytidine deaminase, putative / cytidine aminohydrolase, putative 3.2 AT5G64100 peroxidase, putative 2.9 AT2G15120; [AT2G15120, pseudogene, disease-resistance family/fatty acid 2.9 AT2G15220 elongase-related]: [AT2G15220, secretory protein, putative] AT3G13310 DNAJ heat shock N-terminal domain-containing protein 2.8 AT4G16260 glycosyl hydrolase family 17 protein 2.8 AT2G43510 ATTI1 (A.t. TRYPSIN INHIBITOR protein 1) 2.7 AT1G30700 FAD-binding domain-containing protein 2.6 AT4G28530 ANAC074 (Arabidopsis NAC domain containing protein 74); TF 2.6 AT3G45130 LAS1 (Lanosterol synthase 1); catalytic/ lyase 2.6 AT2G44130; [AT2G44130, kelch repeat-containing F-box]; 2.6 AT2G44140 [AT2G44140, Autophagy 4a] AT3G21850 ASK9 (ARABIDOPSIS SKP1-LIKE 9); ubiquitin-protein ligase 2.5 AT3G09220 LAC7 (laccase 7); copper ion binding / oxidoreductase 2.5 AT3G29250 oxidoreductase 2.5 AT3G21840 ASK7 (ARABIDOPSIS SKP1-LIKE 7); ubiquitin-protein ligase 2.5 AT5G25820 exostosin 2.5 AT2G43570 chitinase, putative 2.4 AT2G29350 SAG 13 (Senescence-associated gene 13); oxidoreductase 2.4 AT2G22470 AGP2 (ARABINOGALACTAN-protein 2) 2.4 AT4G25000 AMY1 (ALPHA-AMYLASE-LIKE); alpha-amylase 2.4 AT5G66690 UDP-glucoronosyl/UDP-glucosyl transferase 2.3 AT4G21680 proton-dependent oligopeptide transport (POT) 2.3 AT4G12550 AIR1 (Auxin-Induced in Root cultures 1); lipid binding 2.3 AT4G38620 MYB4 (myb domain protein 4); transcription factor 2.3 AT4G24000 ATCSLG2 (Cellulose synthase-like G2); glycosyl transferase 2.3 AT3G13610 oxidoreductase, 20G-Fe(ll) oxygenase 2.3 AT3G04720 PR4 (PATHOGENESIS-RELATED 4) 2.2 AT5G40730 AGP24 (ARABINOGALACTAN protein 24) 2.2 AT5G06330 harpin-responsive protein, putative (HIN1) 2.2 AT5G53870 plastocyanin-like domain-containing protein 2.2 AT5G54040 DC1 domain-containing protein 2.2 138 Appendix C (Continued) AT3G20960 CYP705A33 cytochrome P450; oxygen binding 2.2 AT4G22070 WRKY31 (WRKY DNA-binding protein 31); transcription factor 2.1 AT1G67330 similar to unknown protein AT1G27930.1; contains DUF579 2.1 AT4G29570 cytidine deaminase/C95cytidine aminohydrolase, putative 2.1 AT1G68795 CLE12 (CLAVATA3/ESR-RELATED 12); receptor binding 2.0 AT3G51860 CAX3 (cation exchanger 3); cation:cation antiporter 2.0 AT1G22440 alcohol dehydrogenase, putative 2.0 AT1G51860 leucine-rich repeat protein kinase, putative 2.0 AT5G55450 protease inhibitor/seed storage/lipid transfer protein (LTP) 2.0 AT1G65610 endo-1,4-beta-glucanase, putative / cellulase, putative 2.0 AT5G63600 flavonol synthase, putative 2.0 AT5G24210 lipase class 3 2.0 AT3G12700 aspartyl protease 2.0 AT3G44320 NIT3 (NITRILASE 3) 2.0 AT5G42860 similar to unknown protein AT 1G45688 2.0 AT2G44790 UCC2 (UCLACYANIN 2); copper ion binding 2.0 AT5G43350; [AT5G43350, PHT 1; carbohydrate/phosphate/sugar porter]; 2.0 AT5G43370 [AT5G43370, PHT2 (phosphate transporter 2)] AT4G30170 peroxidase, putative 2.0 AT2G38380; [AT2G38380, peroxidase 22]; 2.0 AT2G38390 [AT2G38390, peroxidase, putative] AT3G22800 leucine-rich repeat / extensin 1.9 AT2G01890 Purple Acid Phosphatase 8 1.9 AT4G37010 caltractin, putative / centrin, putative 1.9 AT5G17330 GAD (Glutamate decarboxylase 1); calmodulin binding 1.9 AT2G04160 AIR3 (Auxin-Induced in Root cultures 3); subtilase 1.9 AT5G63970 copine-related 1.9 AT3G54590 ATH RG P1; structural constituent of cell wall 1.9 AT5G64120 peroxidase, putative 1.9 AT3G18280 protease inhibitor/seed storage/lipid transfer protein (LTP) 1.9 AT5G40780 LHT1 (Lys- His TRANSPORTER 1); amino acid permease/transporter 1.9 AT5G24090 acidic endochitinase (CHIB1) 1.9 f,-rr>noRA-rc\ similar to ADR1-LIKE 1, ATP/protein binding, probable disease resistance 1.9 protein At4g33300; contains GHMP Kinase, C-terminal domain AT3G12500 ATHCHIB (BASIC CHITINASE) 1.9 AT 1G29160 Dof-type zinc finger domain-containing protein 1.9 AT4G36990 HSF4 (HEAT SHOCK FACTOR 4); DNA binding / transcription factor 1.8 ATI G17860 trypsin and protease inhibitor / Kunitz family 1.8 AT4G23700 ATCHX17 (CATION/H+ EXCHANGER 17) 1.8 AT2G38870 protease inhibitor, putative 1.8 AT3G04000 short-chain dehydrogenase/reductase (SDR) 1.8 AT5G06720 peroxidase, putative 1.8 AT3G53480 PLEIOTROPIC DRUG RESISTANCE 9; ATPase 1.8 ATI G74020 SS2 (STRICTOSIDINE SYNTHASE 2) 1.8 AT4G30280 XTH18 xyloglucan:xyloglucosyl transferase; glyeosyl hydrolase 1.8 AT4G30670 unknown protein 1.8 AT1G75900 family II extracellular lipase 3 (EXL3) 1.8 AT 1G79450 LEM3 (ligand-effect modulator 3) I CDC50 1.8 AT1G10550 XTH33 (xyloglucan:xyloglucosyl transferase 33); glyeosyl hydrolase 1.8 139 Appendix C (Continued) AT3G14680 CYP72A14 cytochrome P450; oxygen binding 1.8 AT3G21560 UGT84A2; UDP-glycosyltransferase/sinapate 1-glucosyltransferase 1.7 AT3G26440 similar to unknown protein AT 1G13000; contains DUF707 1. AT2G02990 RNS1 (RIBONUCLEASE 1); endoribonuclease 1. AT2G05940 protein kinase, putative 1. AT4G22460 protease inhibitor/seed storage/lipid transfer protein (LTP) 1. AT3G18250 unknown protein 1. AT3G03520 phosphoesterase 1. AT5G19875 similar to oxidoreductase/transition metal ion binding AT2G31940 1. AT4G39270 leucine-rich repeat transmembrane protein kinase, putative 1. AT2G21180 similar to unknown protein AT5G19875 1. AT4G22470 protease inhibitor/seed storage/lipid transfer protein (LTP) 1. AT1G05710 ethylene-responsive protein, putative 1. AT3G54040 photoassimilate-responsive protein-related 1. AT2G19570 CDA1 (CYTIDINE DEAMINASE 1) 1. AT4G23680 major latex protein-related / MLP-related 1. AT4G00700 C2 domain-containing protein 1. AT3G04510 sim. to Light-dep. Short Hypocotyls 1 & to Rubisco, large chain; DUF640 1. AT4G30590 plastocyanin-like domain-containing protein 1. AT2G23620 esterase, putative 1. AT5G64110 peroxidase, putative 1. AT3G49120; [AT3G49120, PEROXIDASE 34]; AT3G49110 [AT3G49110, PEROXIDASE 33] " AT5G13930 CHS (CHALCONE SYNTHASE); naringenin-chalcone synthase 1. AT5G48290 heavy-metal-associated domain-containing protein 1. AT4G23690 disease resistance-responsive family / dirigent family 1. AT5G38030 MATE efflux 1. AT4G32870 similar to unknown protein AT2G25770; contains domain Bet v1-like 1. AT3G48920 AtMYB45 (myb domain protein 45); DNA binding / transcription factor 1.6 AT5G38780 S-adenosyl-L-methionine:carboxyl methyltransferase 1.6 AT1G69880 ATH8 (thioredoxin H-type 8); thiol-disulfide exchange intermediate 1.6 AT3G50570 hydroxyproline-rich glycoprotein 1.6 AT1G09560 Germin-like protein 5; manganese ion/metal ion binding, nutrient reservoir 1.6 AT 1G65500 similar to unknown protein AT 1G65490.1 1.6 AT4G20110 vacuolar sorting receptor, putative 1.6 AT5G18840 sugar transporter, putative 1.6 AT5G44380 FAD-binding domain-containing protein 1.6 AT2G17500 auxin efflux carrier 1.6 AT3G22060 receptor protein kinase-related 1.6 AT5G01050; [AT5G01050, laccase/diphenol oxidase]; AT5G01040 [AT5G01040, LAC8(laccase8)] AT4G25810 XTR6 (Xyloglucan Endotransglycosylase 6); glycosylhydrolase 1.6 AT2G29995 unknown protein 1.6 AT3G62270 anion exchange 1.6 AT4G16240 similar to glycine-rich protein AT5G46730 & AT2G05440 1.6 AT1G63840 zinc finger (C3HC4-type RING finger) 1.6 AT4G26150 zinc finger (GATA type) 1.6 AT4G25900 aldose 1-epimerase 1.6 AT4G18360 peroxisomal/glycolate oxidase/short chain a-hydroxy acid oxidase, put. 1.6 140 Appendix C (Continued) AT1G79530; [AT1G79530, GAPCP-1; GAP-dehydrogenase]; AT1G16300 [AT1G16300, GAPCP-2] AT4G02280 SUS3; UGT/sucrose synthase 1.6 AT2G32660 disease resistance / LRR 1.6 AT2G16720 MYB7 (myb domain protein 7); DNA binding / transcription factor 1.6 AT4G09500 glycosyltransferase 1.6 AT4G09420 disease resistance protein (TIR-NBS class), putative 1.6 AT5G46140 similarto unknown protein AT5G46130.1; contains DUF295 1.6 AT4G01070 UDP-glucoronosyl/UDP-glucosyl transferase 1.6 AT5G46050 ATPTR3/PTR3(PEPTIDE TRANSPORTER protein 3 1.6 AT 1G63530 similar to hydroxyproline-rich glycoprotein AT 1G63540 1.6 AT1G62510 protease inhibitor/seed storage/lipid transfer protein (LTP) 1.6 AT4G32650 ATKC1 (A.t. K+ RECTIFYING CHANNEL 1); cyclic nucleotide binding 1.6 AT3G46280 protein kinase-related 1.6 AT 1G23800 ALDH2B7 3-chloroallyl aldehyde dehydrogenase (NAD) 1.6 AT2G22170 lipid-associated 1.6 AT2G02850 ARPN (PLANTACYANIN); copper ion binding 1.6 AT3G14360 lipase class 3 1.6 AT5G45380 sodium:solute symporter 1.6 AT1G67360 rubber elongation factor (REF) 1.5 AT1G14210 ribonuclease T2 1.5 AT2G41380 embryo-abundant protein-related 1.5 AT4G01610 cathepsin B-like cysteine protease, putative 1.5 AT3G50480 HR4 (HOMOLOG OF RPW8 4) 1.5 AT1G65510 similar to unknown protein ATI G65490.1 1.5 AT5G36220 CYP81D1 (CYTOCHROME P450 91 Al); oxygen binding 1.5 AT5G43060 cysteine proteinase, putative / thiol protease, putative 1.5 AT4G37310 CYP81 HI cytochrome P450 1.5 AT5G55170 SUM3 (SMALL UBIQUITIN-LIKE MODIFIER 3) 1.5 AT4G37870 phosphoenolpyruvate carboxykinase (ATP) / PEPCK, putative 1.5 AT4G23010 ATUTR2/UTR2 (UDP-GALACTOSE TRANSPORTER 2) 1.5 AT3G02910 similar to unknown protein AT5G46720; contains UPF0131 1.5 AT4G33420 peroxidase, putative 1.5 AT4G21850 methionine sulfoxide reductase domain-containing protein 1.5 AT3G29810 phytochelatin synthetase /COBRA cell expansion protein C0BL2 1.5 AT5G45280 pectinacetylesterase, putative 1.5 AT5G13180 ANAC083 (A.t. NAC domain containing protein 83); transcription factor 1.5 AT1G10150 ATPP2-A10 (Phloem protein 2-A10) 0.7 AT3G61060 ATPP2-A13 0.7 AT5G61440 thioredoxin family 0.7 AT4G39540 shikimate kinase family 0.7 AT3G15070 zinc finger (C3HC4-type RING finger) family 0.7 AT5G22270 similar to unknown protein AT3G11600.1 0.7 AT5G52970 thylakoid lumen 15.0 kDa protein 0.7 AT1G24575 unknown protein 0.7 AT5G27950 kinesin motor protein-related 0.7 AT1G33050 sim. to unknown protein AT4G 10470.1 & to Subtilisin-like serine protease 0.7 AT1G74560 NRP1 (NAP1-RELATED protein 1); DNA/chromatin/histone binding 0.7 141 Appendix C (Continued) AT5G51830 pfkB-type carbohydrate kinase family 0.7 AT3G25910 similar to unknown protein AT3G24740.2; similar to TRAF-like; DUF1644 0.7 AT1G11260 STP1 (SUGAR TRANSPORTER 1); carbohydrate transporter 0.7 AT5G16180 ATCRS1/CRS1 (A.f. ortholog of maize chloroplast splicing factor CRS1) 0.7 AT1G48600 phosphoethanolamine N-methyltransferase 2, putative (NMT2) 0.7 AT5G46710 zinc-binding family 0.7 AT3G45300 IVD (ISOVALERYL-COA-DEHYDROGENASE) 0.7 AT2G39310 jacalin lectin family 0.7 AT4G33666 unknown protein 0.7 AT5G23020 MAM-L, Methylthioalkylmalate synthase-like; 2-isopropylmalate synthase 0.7 AT1G61800 GPT2 (glucose-6-phosphate/phosphate translocator 2); antiporter 0.7 AT4G17490 ATERF6 (Ethylene Responsive Element Binding Factor 6); TF 0.7 AT1G21100 O-methyltransferase, putative 0.7 AT5G52310 COR78 (COLD REGULATED 78) 0.7 AT1G15940 similar to binding AT1G80810; contains ARM repeat 0.7 AT1G64490 sim. to unkn. protein AT5G42060 & to putative transcriptional coactivator 0.7 AT4G19170 NCED4 (NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 4) 0.7 AT5G26740 similar to unknown protein AT3G05940.1; contains DUF300 0.6 AT5G20150 SPX (SYG1/Pho81/XPR1) domain-containing protein 0.6 AT2G41640 sim. to unknown protein AT3G57380.1 & to glycosyltransferase; DUF563 0.6 AT1G03090 MCCA (3-methylcrotonyl-CoA carboxylase 1) 0.6 AT5G57630 CIPK21 (CBL-INTERACTING protein KINASE 21); kinase 0.6 AT4G28040 nodulin MtN21 family 0.6 AT5G57340 similar to unknown protein AT5G67390.2 0.6 AT5G24770; [AT5G24770, VSP2 (Vegetative Storage Protein 2); acid phosphatase]; AT5G24780 [AT5G24780, VSP1; acid phosphatase] ° ® AT4G20820 FAD-binding domain-containing protein 0.6 AT1G70290 ATTPS8 {A.t. trehalose phosphatase/synthase 8); glycosyl-transferring 0.6 AT5G06560 similar to unknown protein AT3G11850.2; contains DUF593 0.6 AT2G34620 mitochondrial transcription termination factor-related/mTERF-related 0.6 AT4G26260 MI0X4 (MYO-INOSITOL OXYGENASE 4) 0.6 AT3G22420 WNK2 (WITH NO K 2); kinase 0.6 AT1G08630 THAI (THREONINE ALDOLASE 1); aldehyde-lyase 0.6 AT4G17460 HAT1 (homeobox-leucine zipper protein 1); transcription factor 0.6 AT 1G72070; [AT 1G72070, DNAJ heat shock N-terminal domain-containing protein]; AT1G72060 [AT1G72060, serine-type endopeptidase inhibitor] AT4G19160 binding 0.6 AT3G62950 glutaredoxin family 0.6 AT4G00780 meprin and TRAF homology/MATH domain-containing protein 0.6 AT5G55300 TOPI BETA (DNA TOPOISOMERASE 1 BETA); type I 0.6 AT4G01560 MEE49 (maternal effect embryo arrest 49) 0.6 AT1G10070 ATBCAT-2; branched-chain-amino-acid transaminase/catalytic 0.6 AT1G02640 BXL2 (BETA-XYLOSIDASE 2); 0-glycosyl hydrolase 0.6 AT1G35140 PHI-1 (PHOSPHATE-INDUCED 1) 0.6 AT1G48160 signal recognition particle 19 kDa protein, putative/SRP19, putative 0.6 AT3G44630 disease resistance protein RPP1-WsB-like (TIR-NBS-LRR class), putative 0.6 AT4G10120 ATSPS4F; sucrose-phosphate synthase/transferase 0.6 AT3G49780 ATPSK4 (PHYTOSULFOKINE 4 PRECURSOR); growth factor 0.6 AT2G22990 SNG1 (SINAPOYLGLUCOSE 1); serine carboxypeptidase 0.6 142 Appendix C (Continued) AT1G17280; [AT1G17280, UBC34 ubiquitin-conjugating enzyme; ubiquitin-protein AT5G50430 ligase]; [AT5G50430, UBC33; ubiquitin-protein ligase] 0.6 AT 1G64660 ATMGL; catalytic/ methionine gamma-lyase 0.6 AT2G18300 basic helix-loop-helix (bHLH) family 0.6 AT2G42530 cold-responsive/cold-regulated protein (cor15b) 0.6 AT1G29395 COR414-TM1 (cold regulated 414 thylakoid membrane 1) 0.6 AT4G33070 pyruvate decarboxylase, putative 0.6 AT1G21910 AP2 domain-containing transcription factor family 0.6 AT3G02550 lateral organ boundaries domain protein 41 (LBD41) 0.6 AT2G02710 PAC motif-containing protein 0.6 AT3G21650 Ser/Thr protein phosphatase 2A (PP2A) regulatory subunit B', put. 0.6 AT3G21260 GLTP3 (GLYCOLIPID TRANSFER protein 3) 0.6 AT1G20650 protein kinase 0.6 AT2G22080 sim. to zinc finger protein-related AT5G63740.1; sim. to calcium-binding 0.6 AT1G76590 zinc-binding family 0.6 AT 1G04770 male sterility MSS family 0.6 AT3G52170 DNA binding 0.6 AT4G04630 similar to unknown protein AT4G21970.1; contains DUF584 0.6 AT5G48850 male sterility MSS family 0.6 AT1G53870; [AT1G53870, similar to unknown protein AT1G53890.1]; AT1G53890 [AT1G53890, contains DUF567] 0.6 AT2G41100 TCH3 (TOUCH 3) 0.6 AT1G03610 similar to unknown protein AT4G03420.1; contains DUF789 0.6 AT 1G02660 lipase class 3 0.6 AT2G17550 similar to unknown protein AT2G20240.1 0.6 AT3G52072; [Potential natural antisense gene, locus overlaps with AT3G52070]; AT3G52070 [AT3G52070, similar to hypothetical protein [M. truncatula]] 0.6 AT2G22980 SCPL13; serine carboxypeptidase 0.6 AT4G09890 similar to unknown protein AT5G11970.1 0.6 AT3G45780 PH0T1 (phototropin 1); kinase 0.6 AT1G73600 phosphoethanolamine N-methyltransferase 3, putative (NMT3) 0.6 AT4G33050 EDA39 (embryo sac development arrest 39); calmodulin binding 0.6 AT2G18190 AAA-type ATPase 0.6 AT5G55620 similar to unknown protein AT3G09950.1 0.6 AT5G35490 unknown protein 0.6 AT3G49160 pyruvate kinase 0.6 AT5G39890 similar to unknown protein AT5G15120; DUF1637 & Cupin, RmlC-type 0.6 AT4G29950 microtubule-associated protein 0.6 AT1G76410 ATL8; protein binding/zinc ion binding 0.6 AT3G47340 ASN1 (DARK INDUCIBLE 6) 0.6 AT4G36500 similar to unknown protein AT2G18210.1 0.6 AT2G26530 AR781, similar to calmodulin-binding protein AT2G15760; DUF1645 0.6 AT1G75020 LPAT4; acyltransferase 0.6 AT2G24600 ankyrin repeat family 0.6 AT3G55510 similar to unknown protein AT2G18220.1; contains UPF0120 0.6 AT5G61020 ECT3 (evolutionary conserved C-terminal 3) 0.6 AT3G44970 cytochrome P450 0.6 AT2G03390 uvrB/uvrC motif-containing protein 0.6 AT5G23010 MAM1 (2-isopropylmalate synthase 3); 2-isopropylmalate synthase 0.6 143 Appendix C (Continued) AT3G03020 unknown protein 0.6 AT5G49360 BXL1 (BETA-XYLOSIDASE 1); 0-giycosyi hydroiase 0.6 AT1G80920 J8; heat shock protein binding/unfoided protein binding 0.6 AT4G02800 similar to unknown protein AT5G01970.1 0.6 AT4G12720 AtNUDT7 {A.t. NUDIX HYDROLASE HOMOLOG 7); hydrolase 0.6 AT5G14470 GHMP kinase-related 0.6 AT 1G80840 WRKY40 (WRKY DNA-binding protein 40); transcription factor 0.6 AT4G37370 CYP81D8 cytochrome P450 0.6 AT 1G36370 SHM7 (Serine/glycine hydroxymethyltransferase 7) 0.5 AT3G05900 neurofilament protein-related 0.5 AT5G11010 pre-mRNA cleavage complex-related 0.5 AT5G18060 auxin-responsive protein, putative 0.5 AT3G10040 transcription factor 0.5 AT4G24110 similar to Hypothetical protein [Oryza sativa] 0.5 AT1G14280 PKS2 (PHYTOCHROME KINASE SUBSTRATE 2) 0.5 AT5G22920 zinc finger (C3HC4-type RING finger) 0.5 AT4G27450 sim. to unknown At3g15450; N-terminal nucleophile aminohydrolase dom. 0.5 AT5G41080 glycerophosphoryl diester phosphodiesterase 0.5 AT5G44260 zinc finger (CCCH-type) family 0.5 AT2G26980 CIPK3 (CBL-INTERACTING protein KINASE 3) 0.5 AT4G10270 wound-responsive family 0.5 AT1G35612 pseudogene of Ulpl protease family protein 0.5 AT2G15080 disease resistance family 0.5 AT3G29290 EMB2076 (EMBRYO DEFECTIVE 2076); binding 0.5 AT1G73540 ATNUDT21 {A.t. Nudix hydrolase homolog 21); hydrolase 0.5 AT2G04050 MATE efflux family 0.5 AT2G17850 similar to unknown protein AT5G66170.2; Rhodanese-like domain 0.5 AT1G74670 gibberellin-responsive protein, putative 0.5 AT2G32880; [AT2G32880, MATH domain-containing protein]; 0.5 AT2G32870 [AT2G32870, MATH domain-containing protein] AT2G38470 WRKY33 (WRKY DNA-binding protein 33); transcription factor 0.5 AT 1G05680 UDP-glucoronosyl/UDP-glucosyl transferase family 0.5 AT3G55980 zinc finger (CCCH-type) family 0.5 AT5G47240 ATNUDT8 (A.t. Nudix hydrolase homolog 8); hydrolase 0.5 AT1G64360 unknown protein 0.5 AT5G15960; [AT5G15960, KIN1]; 0.5 AT5G15970 [AT5G15970, KIN2 (COLD-RESPONSIVE 6.6)] AT3G02040 Senescence-Related Gene 3; glycerophosphodiester phosphodiesterase 0.4 AT3G13080; [AT3G13080, ATMRP3 (A.t. multidrug resistance-associated protein 3)]; 0.4 AT1G71330 [AT1G71330, ATNAP5 (A.t. non-intrinsic ABC protein 5)] AT1G33055 unknown protein 0.4 AT2G45660 AGL20 (AGAMOUS-LIKE 20); transcription factor 0.4 AT1G21110; [AT1G21110, 0-methyltransferase, putative]; 0.4 AT1G21120 [AT1G21120, 0-methyltransferase, putative] AT3G23030 IAA2 (indoleacetic acid-induced protein 2); transcription factor 0.4 AT5G46240 KAT1, K+ ATPase; cyclic nucleotide binding/inward rectifier K+ channel 0.4 AT5G56870 beta-galactosidase, putative/lactase, putative 0.4 AT2G22880 VQ motif-containing protein 0.4 AT3G48710 GTP binding/RNA binding 0.3 144 Appendix C (Continued) AT5G66985 unknown protein 0.3 AT5G12050 sim. to unknown prot. AT1G54200 & to putative ATP-dep. DNA helicase 0.3 AT1G76650 calcium-binding EF hand 0.3 145 Appendix D Gene loci identified by microarray as up- or down-regulated by BaP in 24 h Locus ID FC Annotation At4g 17490 28.1 ATERF6 (Ethylene-Responsive binding Factor 6); TF At5g42900 23.6 similar to unknown protein At4g33980 At4g33980 19.1 similar to unknown protein At5g42900 At2g14560 8.1 similarto unknown protein At1g33840; contains DUF567 At1g17665 7.1 BoCAR; similar to CA-responsive protein [S. oleracea] At3g55980 7.1 zinc finger (CCCFI-type) family At5g45340 7.1 CYP707A3 (cytochrome P450) At1g07050 6.9 CONSTANS-like protein-related At2g40080 6.5 ELF4 (EARLY FLOWERING 4) At4g24570 5.6 mitochondrial substrate carrier family At1g27730 4.7 STZ (SALT TOLERANCE ZINC FINGER); TF At3g04640 4.7 glycine-rich protein At5g61600 4.7 ethylene-responsive element-binding family At4g29780 4.6 similarto unknown protein At5g12010 At5g52310 4.4 COR78 (COLD REGULATED 78) At5g23240 4.3 DNAJ heat shock N-terminal domain-containing At1g80840 4.1 WRKY40 (WRKY DNA-binding protein 40); TF At2g40140 4.1 CZF1/ZFAR1; transcription factor At5g48250 4.1 zinc finger (B-box type) family protein At1g05575 3.9 unknown protein At2g40000 3.9 similar to unknown protein At3g55840 At4g27280 3.8 calcium-binding EF hand family At5g51190 3.8 AP2 domain-containing transcription factor, putative At5g57220 3.8 CYP81F2 cytochrome P450 At3g59350 3.7 serine/threonine protein kinase, putative At1g51090 3.6 heavy-metal-associated domain-containing At5g50450 3.6 zinc finger (MYND type) family At3g50930 3.5 AAA-type ATPase family protein At4g11280 3.5 ACS6 (1-ACC SYNTHASE 6) At4g37260 3.5 AtMYB73/MYB73 (myb domain protein 73) At5g61380 3.5 T0C1 (TIMING OF CAB EXPRESSION 1) At1g61890 3.4 MATE efflux family At4g23810 3.4 WRKY53 transcription activator/TF At5g57110 3.4 ACA8 (Autoinhibited Ca2+ -ATPASE, Isoform 8) At4g34131 3.3 UGT73B3; UDP/abscisic acid glucosyltransferase At4g34135 3.3 UGT73B2; UDP/flavonol 3-0-glucosyltransferase At5g 15960 3.3 KIN2 (COLD-RESPONSIVE 6.6) At5g15970 3.3 KIN1 At2g21130 3.2 peptidyl-prolyl cis-trans isomerase / cyclophilin (CYP2) At4g 12280 3.2 copper amine oxidase family At4g12290 3.2 copper amine oxidase family At2g38470 3.1 WRKY33 (WRKY DNA-binding protein 33) At2g41640 3.1 similarto unknown protein At3g57380 At3g05800 3.1 transcription factor At5g04340 3.1 C2H2 zinc finger TF (ZAT6) At1g11210 3 similar to unknown protein A tlg l 1220; contains DUF761 At1g57980 3 purine permease-related At1g57990 3 ATPUP18 (A.t. purine permease 18) At1g76600 3 similar to unknown protein At1g21010 At4g16260 3 glycosyl hydrolase family 17 At4g30650 3 hydrophobic/low temperature & salt responsive prot., putative At5g27420 3 zinc finger (C3HC4-type RING finger) family At5g44420 3 PDF1.2 (Low-molecular-weight cysteine-rich 77) At5g62360 3 invertase/pectin methylesterase inhibitor family At1g68050 2.9 FKFl, Flavin-binding Kelch domain F box; ubiquitin- ligase At1g77450 2.9 ANAC032 (Arabidopsis NAC domain containing TF At4g31800 2.9 WRKY18 transcription factor At5g23410 2.9 similar to FKFl; contains domain PTHR23244 At5g42730 2.9 pseudogene similar to ACT domain-containing, F-box At5g60100 2.9 APRR3 (PSEUDO-RESPONSE REGULATOR 3) At1g 18570 2.8 MYB51 (MYB DOMAIN PROTEIN 51) At1g67970 2.8 AT-HSFA8 transcription factor At4g17500 2.8 ATERF-1, Ethylene Responsive Element binding Factor At1g 17420 2.7 L0X3 (Lipoxygenase 3) At5g11150 2.7 ATVAMP713 (vesicle-associated membrane protein) At5g17340 2.7 similar to unknown protein At3g03272 At5g59550 2.7 zinc finger (C3HC4-type RING finger) family At1g17170 2.6 ATGSTU24 glutathione transferase At1g55450 2.6 embryo-abundant protein-related At2g47890 2.6 zinc finger (B-box type) family At3g08720 2.6 ATPK19 (A.t. PROTEIN KINASE 19); kinase At3g20810 2.6 jumonji OmjC) domain-containing transcription factor At5g10695 2.6 similar to unknown protein At5g57123 At5g57630 2.6 CIPK21 (CBL-INTERACTING PROTEIN KINASE 21) At2g42530 2.5 C0R15B At4g33050 2.5 EDA39 (embryo sac development arrest 39) At1g73540 2.4 ATNUDT21 (A.t. Nudix hydrolase homolog 21) At1g76790 2.4 0-methyltransferase family 2 protein At1g80590 2.4 WRKY66 (WRKY DNA-binding protein 66) At2g38790 2.4 unknown protein At4g01870 2.4 tolB protein-related At4g27560 2.4 glycosyltransferase family protein At4g27570 2.4 glycosyltransferase family protein At4g36010 2.4 pathogenesis-related thaumatin family At5g06320 2.4 NHL3 (NDRI/HINI-like 3) At5g23660 2.4 MTN3 (HOMOLOG OF M. truncatula MTN3) At5g39020 2.4 protein kinase family protein At1g49230 2.3 zinc finger (C3HC4-type RING finger) family At1g72900 2.3 disease resistance protein (TIR-NBS class), putative At1g73500 2.3 ATMKK9 (A.t. MAP kinase kinase 9); kinase At2g16365 2.3 F-box family protein At2g26530 2.3 AR781 At2g31880 2.3 LRR transmembrane protein kinase, putative At3g46620 2.3 zinc finger (C3HC4-type RING finger) family At3g56710 2.3 SIB1 (SIGMA FACTOR BINDING PROTEIN 1) 147 At4g34950 2.3 nodulin family protein At5g22250 2.3 CCR4-N0T transcription complex protein, putative At5g46710 2.3 zinc-binding family protein At1g05560 2.2 UGTl (UDP-glucosyl transferase 75B1) At1g28330 2.2 DRM1 (DORMANCY-ASSOCIATED PROTEIN 1) At1g69890 2.2 similar to unknown protein At1g27100 At2g02390 2.2 ATGSTZ1 (GLUTATHIONE S-TRANSFERASE 18) At2g 19450 2.2 TAGl (Triacylglycerol Biosynthesis Defect 1) At2g26560 2.2 PLP2 (PHOSPHOLIPASE A 2A); nutrient reservoir At2g36790 2.2 UGT73C6 (UDP-GLUCOSYL TRANSFERASE) At2g36970 2.2 UDP-glucoronosyl/UDP-glucosyl transferase At3g23605 2.2 UBX domain-containing protein At4g16146 2.2 similar to unknown protein At1g69510 At5g 18470 2.2 curculin-like (mannose-binding) lectin family At5g60900 2.2 RLK1 (Receptor-Like Protein Kinase 1) At5g67480 2.2 BT4 (BTB and TAZ domain protein 4) At1g11130 2.1 SUB (STRUBBELIG); protein binding All g 12200 2.1 flavin-containing monooxygenase family At1g21250 2.1 WAKl (CELL WALL-ASSOCIATED KINASE) At1g23410 2.1 ubiquitin extension protein, putative; RPS27a At1g55850 2.1 ATCSLE1 (Cellulose synthase-like El) At1g72940 2.1 disease resistance protein (TIR-NBS class), putative At1g73330 2.1 ATDR4 (A.t. drought-repressed 4) At1g76680 2.1 OPRl (12-oxophytodienoate reductase 1) At1g76690 2.1 0PR2 (12-oxophytodienoate reductase 2) At2g36800 2.1 DOGTl (DON-GLUCOSYLTRANSFERASE) At2g42540 2.1 C0R15A (COLD-REGULATED 15A) At3g28290 2.1 AT14A At3g28300 2.1 AT14A At3g59140 2.1 ATMRP14 (multidrug resistance-associated protein) At3g59820 2.1 calcium-binding mitochondrial protein-related At4g36500 2.1 similar to unknown protein At2g18210 At5g24470 2.1 APRR5 (PSEUDO-RESPONSE REGULATOR 5) At5g63790 2.1 ANAC102 transcription factor At1g23830 2 similar to unknown protein At1g23840 At1g27770 2 ACA1 (autoinhibited Ca2+ -ATPase 1) At2g21660 2 ATGRP7 (Cold, Circadian Rhythm, & RNA binding 2) At2g22450 2 riboflavin biosynthesis protein, putative At3g16530 2 legume lectin family protein At3g28740 2 cytochrome P450 family protein At4g19120 2 ERD3 (EARLY-RESPONSIVE TO DEHYDRATION 3) At4g19880 2 similar to unknown protein At5g45020 At5g23050 2 acyl-activating enzyme 17 (AAE17) At5g39410 2 binding / catalytic At5g54960 2 PDC2 (PYRUVATE DECARBOXYLASE-2) At2g07675 1.9 ribosomal protein SI 2 mitochondrial family ATMG00980 1.9 ribosomal protein L2 At1g02340 0.6 HFR1 (LONG HYPOCOTYL IN FAR-RED) A tig 10370 0.6 GSTU17/GST30/Early-Responsive to Dehydration 9 148 At1g34310 0.6 ARF12 (AUXIN RESPONSE FACTOR 12) At1g64670 0.6 BDG1 (B0DYGUARD1); hydrolase At2g21560 0.6 similar to unknown protein At4g39190 At3g21670 0.6 nitrate transporter (NTP3) At3g28270 0.6 similar to A T U A At3g28290 & At3g28300; DUF677 At4g30610 0.6 BRS1 (BRI1 SUPPRESSOR 1) At4g38850 0.6 SAUR_AC1 (SMALL AUXIN UP RNA 1) At5g02670 0.6 similar to poly(A)transferase/protein binding At3g06560 At5g59780 0.6 MYB59 (myb domain protein 59) At1g07180 0.5 ATNDI1/NDA1 (Alternative NAD(P)H Dehydrogenase 1) At1g07350 0.5 transformer serine/arginine-rich ribonucleoprotein, putative At1g 13080 0.5 CYP71B2 CYTOCHROME P450 A tig 14280 0.5 PKS2 (PHYTOCHROME KINASE SUBSTRATE 2) At1g29430 0.5 auxin-responsive family protein At1g32450 0.5 proton-dependent oligopeptide transport (POT) family At1g44000 0.5 similar to unknown protein At4g11911 At1g62510 0.5 protease inhibitor/seed storage/lipid transfer protein (LTP) At1g62960 0.5 ACS10 (ACC SYNTHASE 10) At1g75180 0.5 similar to unknown protein At1g19400 At1g79270 0.5 ECT8 (evolutionarily conserved C-terminal region 8) At2g 16370 0.5 THY-1 (THYMIDYLATE SYNTHASE 1) At2g 17830 0.5 F-box family protein At2g21185 0.5 unknown protein At2g21187 0.5 Potential natural antisense gene, overlaps with At2g21185 At2g26690 0.5 nitrate transporter (NTP2) At2g30520 0.5 RPT2 (ROOT PHOTOTROPISM 2); protein binding At2g31380 0.5 STH (salt tolerance homologue); zinc ion binding TF At2g32530 0.5 ATCSLB03 (Cellulose synthase-like B3) At2g32540 0.5 ATCSLB04 (Cellulose synthase-like B4) At2g37170 0.5 PIP2B (plasma membrane intrinsic protein 2;2) At2g37180 0.5 RD28 (plasma membrane intrinsic protein 2;3) At2g42190 0.5 sim. to unknown protein At3g57930; HMG-1 & HMG-Y domain At2g43620 0.5 chitinase, putative At2g45660 0.5 AGL20 (AGAMOUS-LIKE 20); transcription factor At3g01550 0.5 those phosphate/phosphate translocator, putative At3g11770 0.5 nucleic acid binding At3g12580 0.5 HSP70 (heat shock protein 70); ATP binding At3g14770 0.5 nodulin MtN3 family protein At3g15310 0.5 transposable element gene At3g26310 0.5 CYP71B35 cytochrome P450 At3g27170 0.5 CLC-B (chloride channel protein B) At3g48100 0.5 ARR5 (A.t. Response Regulator 5) At3g50560 0.5 short-chain dehydrogenase/reductase (SDR) family At3g54720 0.5 AMP1 (Altered Meristem Program 1); dipeptidase At3g55920 0.5 peptidyl-prolyl cis-trans isomerase/cyclophilin, putative At3g61890 0.5 ATHB-12 (A.t. HOMEOBOX PROTEIN 12) At3g63200 0.5 PLA IIIB/PLP9 (Patatin-like protein 9) At4g 12980 0.5 auxin-responsive protein, putative At4g22470 0.5 protease inhibitor/seed storage/lipid transfer protein (LTP) 149 At4g22570 0.5 APT3 (Adenine phosphoribosyltransferase 3) At4g23290 0.5 protein kinase family protein At4g34570 0.5 THY-2 (THYMIDYLATE SYNTHASE 2) At4g38860 0.5 auxin-responsive protein, putative At5g12050 0.5 similar to unnamed protein [\/. vinifera] (GB:CA045643.1) At5g12110 0.5 elongation factor IB alpha-subunit 1 (eEF1Balpha1) At5g27780 0.5 auxin-responsive family protein At5g35970 0.5 DNA-binding protein, putative At5g42460 0.5 F-box family protein At5g47370 0.5 HAT2; transcription factor At5g48490 0.5 LTP At5g64840 0.5 ATGCN5 (A.t. general control non-repressible 5) At5g64940 0.5 ATATH13 (ABC2 homolog 13) At5g66590 0.5 allergen V5/Tpx-1-related family protein At1g02820 0.4 late embryogenesis abundant 3 family / LEA3 family At1g 12650 0.4 contains DUF947 (lnterPro:IPR009292) At1g29460 0.4 auxin-responsive protein, putative At1g68190 0.4 zinc finger (B-box type) family protein At1g69160 0.4 unknown protein At1g69530 0.4 ATEXPA1 (A.t. EXPANSIN A l) At1g74670 0.4 gibberellin-responsive protein, putative At1g75100 0.4 JAC1; HSP binding At1g76110 0.4 high mobility group (HMG1/2), ARID/BRIGHT domain At2g39705 0.4 DVL11/RTFL8 (ROTUNDIFOLIA LIKE 8) At2g40610 0.4 ATEXPA8 (A.t. EXPANSIN A8) At3g04810 0.4 ATNEK2; kinase At3g14200 0.4 DNAJ heat shock N-terminal domain-containing protein At3g17510 0.4 CIPK1 (CBL-INTEFtACTING PROTEIN KINASE 1) At3g47340 0.4 ASN1 (DARK INDUCIBLE 6) At3g54500 0.4 similar to dentin sialophosphoprotein-related At5g64170 At3g57040 0.4 ARR9 (RESPONSE REACTOR 4); transcription regulator At5g35490 0.4 unknown protein At5g62280 0.4 similar to unknown protein At2g45360; contains DUF1442 At1g 13650 0.3 similar to gar2-related At2g03810 At1g55960 0.3 similar to unknown protein At3g13062; Lipid-binding START At1g64500 0.3 glutaredoxin family protein At2g 15020 0.3 similar to unknown protein At5g64190 At2g41250 0.3 haloacid dehalogenase-like hydrolase family At2g46830 0.3 CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) At3g24500 0.3 MBF1C (Multiprotein Bridging Factor 1c) At4g25100 0.3 FSD1 (FE SUPEROXIDE DISMUTASE 1) At5g06980 0.3 similar to unknown protein At3g 12320 At5g 15850 0.3 C0L1 (CONSTANS-LIKE 1); zinc ion binding TF At5g52900 0.3 similar to unnamed protein [V. vinifera] (GB:CA049548.1) At1g73870 0.2 zinc finger (B-box type) family At2g46670 0.2 pseudo-response regulator/TOCI-like protein, putative At2g46790 0.2 APRR9 (PSEUDO-RESPONSE REGULATOR 9) At3g02380 0.2 COL2 (CONSTANS-LIKE 2); zinc ion binding TF At3g12320 0.2 similar to unknown protein At5g06980 150 Appendix E Primers used in qRT-PCR Primer Locus nt Sequence nM ABC-F At3g59140 23 5'-GTTGTTGGTTACACATCAAGTGG-3' 200 ABC-R At3g59140 23 5'-GATTTCTCCATCTGACATCAACA-3' 200 ACTIN2-F At3g 18780 20 5’-CGCTCTTTCTTTCCAAGCTC-3' 500 ACTIN2-R At3g18780 20 5'-CCATACCGGTACCATTGTCA-3' 500 At14a-F At3g28300 20 5'-TTGAATGCTGTGAAGGATGC-3' 240 At14a-R At3g28300 22 5'-CCGTAAGGGTTTCCATTACTTG-3’ 240 BCAT3-F At3g49680 20 5'-TCCATCAAGCTTCAGCATTC-3' 400 BCAT3-R At3g49680 20 5'-AAACAGCGTTGCATCGAGTA-3' 400 BoCAR-F A tig 17665 23 5'-CAAAGGATTGACACTAGCTCAGG-3' 125 BoCAR-R A tig 17665 22 5-TGCGCTTGATTACTTCTGACTT-3' 125 DIB-F At5g12050 20 5'-CCAAAAGAAACCCGTCATTC-3' 125 DIB-R At5g12050 20 5'-TTGGGTTTTAGGATCGATGG-3' 125 D0X1-F At3g01420 21 5’-CCGTCGATCAGAAATCAAAGT-3' 500 D0X1-R At3g01420 25 5'-TTTTTCCTTCCTAATAGTTTTGTCG-3' 500 E1-F At1g13650 20 5'-CTCTCCCAATACTGCAATCG-3' 500 E1-R At1g13650 21 5'-TCGAACTTTTCTCTCCTCAGC-3' 500 E4-F At4g33980 20 5'-CGAAACCAAACACTCAGAGG-3' 400 E4-R At4g33980 20 5'-TTTGCTCACCTCTCCCTTCT-3' 400 E5-F At5g42900 21 5’-TCTGGCTCAGCCTCTAGTCTC-3' 400 E5-R At5g42900 20 5'-CGATACCTCTGCTTCTCCAA-3' 400 FKF1-F At1g68050 19 5'-CGTTCATTGTTTCCGATGC-3' 125 FKF1-R At1g68050 21 5'-TTTGAGCTCGAGGATCTCTGT-3' 125 GDE-F At5g41080 20 5’-GCAGCCATTAGCAAGATCAA-3' 400 GDE-R At5g41080 20 5'-ACTGCCTCTCCGACATTGTT-3' 400 GH17-F At4g 16260 26 5'-TGATATGACTTTGATTGGAAACTCTT-3' 200 GH17-R At4g16260 22 5’-AACGCTGAGTTCGTACTCGTAA-3' 400 GRP7-F At2g21660 19 5'-GTATCGGTGCTTCGTTGGA-3' 500 GRP7-R At2g21660 24 5'-AATGATCTTGGAATCAATAACGTC-3' 500 hp-F At4g30650 20 5'-ACGTGGCTGTTGCACTGTAG-3' 400 hp-R At4g30650 20 5'-AGAGCCTTCACGGTTTTGAA-3' 400 JMJ-F At3g20810 23 5'-TCCAATGGAGCCTACTTATCTTG-3' 125 JMJ-R At3g20810 22 5'-CCGACAAAACAGTAATCAGGAA-3' 125 LAC7-F At3g09220 20 5’-CAATTCCGACGCTTATACGA-3' 300 LAC7-R At3g09220 22 5'-GGCTAAACATTCTGTCTTTGGA-3' 300 MRP3-F At3g13080 21 5'-CACTGCTGGTTACAAGACTGC-3' 250 MRP3-R At3g13080 20 5'-GGATTGGTCGGTAGAAGCTC-3' 250 MYB4-F At4g38620 20 5'-CTATCTCCGGCCTGACCTTA-3' 500 MYB4-R At4g38620 20 5'-GGCAATAAGCGACCATTTGT-3' 500 NAP-F At4g04460 22 5'-CTTATGCTGCTGAGCTATGTGA-3' 500 NAP-R At4g04460 20 5’-CTTCTGCCACCAATTGAGAA-3’ 500 NUDT7-F At4g12720 21 5'-TGGAGAGAAGAGGGGAAGAAG-3' 250 NUDT7-R At4g12720 20 5'-TCCGCGTGGTGATATCT AAA-3' 250 P450-F At3g28740 22 5'-ACTCTCAACATGGGTTTGTGAA-3' 500 P450-R At3g28740 21 5'-GTCGTTCTCTGTTCCATCACC-3’ 500 151 PRX3a-F At5g64100 19 5'-TGTTGGCGGACACACAATA-3' 500 PRX3a-R At5g64100 19 5'-TCGATTGATGGGTCAGGTT-3' 500 RD29A-F At5g52310 22 5'-GCACCAGGCGTAACAGGTAAAC-3' 250 RD29A-R At5g52310 21 5'-AAACACCTTTGTCCCTGGTGG-3' 250 SS-F At1g74010 19 5’-CCCTGAATCTTTCGCCTTT-3' 500 SS-R At1g74010 18 5'-TGCCGAGTTCGAGGAATC-3' 500 TAZ-F At5g67480 22 5'-CCGTTTCCTCTACTCTTCTTGC-3' 200 TAZ-R At5g67480 19 5'-GCCATTCACAGACCCGTTT-3' 200 T0C1-F At5g61380 21 5'-CCAGGAAAATGAGTGGTCTGT-3' 500 T0C1-R At5g61380 20 5'-GATGATCCAATGGAGGCTCT-3' 500 Tub-F At5g23860 19 5'-ACTGCTTGCAAGGATTCCA-3’ 300 Tub-R At5g23860 19 5'-TCAACAACGTTCCCATTCC-3’ 400 UGT1-F At1g05560 20 5'-GAACGCGAAGCTACTGGAAG-3' 400 UGT1-R At1g05560 21 5'-CACCAAACCATCCTTGTTCTC-3' 400 VAMP713-F At5g11150 21 5'-GCTCGTCGTTACAGAACCATT-3’ 200 VAMP713-R At5g11150 26 5'-CCAAAGCTAGTACAAGTATCAGAGCA-3' 200 WAK1-F At1g21250 20 5'-GTTGCCAAGACGTCAATGAG-3' 250 WAK1-R At1g21250 20 5'-GAAGCCTCCAACCTTGTTTC-3' 250 YLS-F At5g08290 19 5'-GCAGATGGATGAGGTGCTT-3' 250 YLS-R At5g08290 22 5'-CCTGAAGAAGAACATGACCGTA-3' 250 Note: All amplicons are between 60 and 140 nt. 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