Analysis of Tardigrade Damage Suppressor Protein (Dsup) Expressed in Tobacco

by

Justin Kirke

A Thesis Submitted to the Faculty of

The Charles E. Schmidt College of Science

In Partial Fulfillment of the Requirements for the Degree of

Master of Science

Florida Atlantic University

Boca Raton, FL

December 2019 Copyright 2019 by Justin Kirke

ii

Abstract

Author: Justin Kirke

Title: Analysis of Tardigrade Damage Suppressor Protein (Dsup) Expressed in Tobacco

Institution: Florida Atlantic University

Thesis Advisor: Dr. Xing-Hai Zhang

Degree: Master of Science

Year: 2019

DNA damage is one of the most harmful stress inducers in living organisms.

Studies have shown that exposure to high doses of various types of radiation cause DNA sequence changes (mutation) and disturb protein synthesis, hormone balance, leaf gas exchange and enzyme activity. Recent discovery of a protein called Damage Suppressor

Protein (Dsup), found in the tardigrade species Ramazzotius varieornatus, has shown to reduce the effects of radiation damage in human cell lines. We have generated multiple lines of tobacco plants expressing the Dsup gene and preformed numerous tests to show viability and response of these transgenic plants when exposed to mutagenic chemicals,

UV radiation and ionizing radiation. We have also investigated Dsup function in association to DNA damage and repair in plants by analyzing the expression of related genes using RT-qPCR. We have also analyzed DNA damage from X-ray and UV treatments using an Alkaline Comet Assay. This project has the potential to help generate plants that are tolerant to more extreme stress environments, particularly DNA damage and

iv mutation, unshielded by our atmosphere. The possibility of growing plants accompanying human space travel and extraterrestrial colonization inspires our imagination.

Extremotolerant tardigrade genes such as Dsup may be a valuable avenue in helping to cultivate crops in these future endeavors.

v Acknowledgements

I would like to start by acknowledging my thesis advisor and mentor Dr. Xing-Hai

Zhang. Without his insight and guidance, I would not be where I am today. I would also like to thank my other committee members Dr. Mary Jane Sunders and Dr. David

Binninger for their additional guidance and thoughtful input into my project.

I would also like to thank my coworkers and students who volunteered to help during the completion of this work. My fellow lab mates, Paveena Vichyavichien, and

Xiao-Lu Jin. My direct independent research students, Tahoe Albergo, Andrew Balsamo,

Nicholas Nifakos, Milove Jeannot, Amanda Lam, Ronscardy Mondesir, Andrew Adeyiga,

Mohamed Abutineh, and Nicholas Pizzo.

Next, I would like to thank the Department of Biology, the college of medicine, and the FAU high school with all of their help to complete this achievement, including technical support and allowing me the use of equipment require to conduct my study.

I would like to thank my friends and family. Without the emotional support and encouragement of my parents and my life partner Jennifer Castignoli, I would not be where

I am at today.

Lastly, I would also like to thank my scholarly Dungeons and Dragons crew, Amy

Makler, Josh Disatham, Douglas Holmes, Gurtejpal Ghuman, Sean Paz, Anthony

Muscarella, and Erick España for helping distract me from the stress of this journey with another one.

vi Analysis of Tardigrade Damage Suppressor Protein (Dsup) Expressed in Tobacco

List of Tables ...... ix

List of Figures ...... x

Introduction ...... 1

DNA Damage and Repair in plants ...... 1

Damage Suppressor Protein (Dsup) from Tardigrade ...... 3

Single Cell Gel Electrophoresis (Alkaline Comet Assay) ...... 4

Dsup expression in plants ...... 5

Methods and Materials ...... 6

Generation of Transgenic Plants ...... 6

Growth Analysis ...... 8

Histochemical Staining analysis ...... 8

DNA and RNA extraction, cDNA synthesis, and qPCR analysis ...... 8

Analysis of DNA Damage Targets through RT-qPCR (Damage Assay) ...... 9

Single Cell Gel Electrophoresis (Alkaline Comet Assay) ...... 10

Results ...... 12

Bioinformatics Analysis...... 12

Genotyping and Expression of Dsup Transgenic Plants ...... 14

Histochemical Staining analysis ...... 15 vii Growth Assay...... 16

Analysis of DNA Damage Targets by RT-qPCR (Damage Assay) ...... 19

Single Cell Gel Electrophoresis (Alkaline Comet Assay) ...... 27

Discussion ...... 31

References ...... 36

viii List of Tables

Table 1: Target genes tested for expression level changes in Dsup vs Control plants

treated with genomutagens. ↓ showed downregulation, ↑ showed upregulation,

and ≌ showed no change in gene expression...... 20

ix List of Figures

Figure 1: Engineered Ti plasmid containing Dsup and NPTII genes driven by NOS

and CaMV-35S promoters...... 7

Figure 2: Amino acid sequence for Dsup protein based off ascension “P0DOW4”.

Highlighted in yellow is the N-terminus, underlined is the α-helical region, and

highlighted in green is the C-terminus. Boxed in Red is a possible nuclear

localization signal (NLS) ...... 12

Figure 3:I-TASSER Predicted 3D structure of the Dsup Protein ...... 13

Figure 4: pBlast results for α-helical region of the Dsup protein ...... 13

Figure 5: Relative gene dosage of Dsup gene for T0 plants based on reference gene

EFα...... 14

Figure 6: Relative expression of Dsup mRNA in T0 plants based on reference gene

EFα...... 14

Figure 7: Relative gene dosage of Dsup gene for T1 plants based on reference gene

EFα...... 15

Figure 8: GUS stain analysis of T0 tissue regeneration on EMS media...... 16

Figure 9: Average fresh weight of EMS treated samples. *Significant difference

found between treatment samples with p-value < 0.05. p-value = 0.0001262 ...... 17

Figure 10: Average root length of EMS treated samples. *Significant difference

found between treatment samples with p-value > 0.05. p-value = 7.465e-05...... 18

x Figure 11: Growth assay plates for RM1 under 0 mM, 1 mM and 3 mM EMS

treatment...... 19

Figure 12: Growth assay plates for R5-3 under 0 mM, 1 mM and 3 mM EMS

treatment...... 19

Figure 13: Relative gene expression of ATR. Example of upregulation between

treated samples...... 21

Figure 14: Relative gene expression of TGA. Example of downregulation between

treated samples...... 21

Figure 15:Relative gene expression of CASP5/6. Example of downregulation to

normal levels between treated samples...... 22

Figure 16: Relative gene expression of Ku70. Example of no change in regulation

between treated samples...... 22

Figure 17: Relative gene expression of ERCC. Example of downregulation to normal

levels between treated samples...... 23

Figure 18: Relative gene expression of TGA. Example of downregulation to normal

levels between treated samples...... 23

Figure 19: Relative gene expression of RAD51. Example of upregulation between

treated samples...... 24

Figure 20: Relative gene expression of DDB. Example of no change in regulation

between treated samples...... 24

Figure 21: Relative gene expression of ATM. Example of downregulation to normal

levels between treated samples...... 25

xi Figure 22: Relative gene expression of ATR. Example of upregulation between

treated samples...... 25

Figure 23: Relative gene expression of TGA. Example of upregulation between

treated samples...... 26

Figure 24: Relative gene expression of CEN2. Example of no change in regulation

between treated samples...... 26

Figure 25: Alkaline comet assay images for RM1 samples...... 27

Figure 26: Alkaline comet assay images for R5-3 samples...... 28

Figure 27: Average tail movement for UV-C treated samples. *Significant difference

found between treatment samples with p-value < 0.05. p-value = 2.2e-16 ...... 28

Figure 28: Average tail movement for X-ray treated samples. *Significant difference

found between treatment samples with p-value < 0.05. p-value = 9.221e-09 ...... 29

xii Introduction

DNA Damage and Repair in plants

Mechanisms to repair DNA damage are important to the health, growth and development of any plant. While plants have many mechanisms to protect themselves from damage to the genome, it is still possible to cause significant changes in protein synthesis and disrupt natural development [1]. Most natural genome damage in plants can be attributed to two common sources. The sun, in the form of ultraviolet (UV) radiation, and from oxygen, creating reactive oxygen species (ROS) [2][3]. Additionally, anthropogenic or naturally occurring ionizing radiation and alkylation damage from mutagenic chemicals play an important role in creating DNA mutations [4][5].

UV-induced damage is a common issue when considering damage to epidermal tissue in plants and animals. The most common forms from the sun that can reach the earth surface, UV-A (wavelength 320 – 400 nm) and UV-B (280 – 320 nm), can penetrate our atmosphere. The more intense UV-C (< 280 nm) is unable to penetrate and is less of a threat to epidermal cells in an organism. UV-induced DNA damage creates pyrimidine dimers which lead to the halt of DNA replication and transcription. The dimers can hinder the progress of DNA polymerase and RNA polymerase II, causing a complete halt in transcription of affected genes. Since sunlight containing UV-A and UV-B is required for the development of plants, they have adapted by creating flavonoid pigments that effectively absorb this UV radiation. In plants this damage can also be reversed by means of direct DNA damage repair known as photoreactivation. The photolyase enzyme directly

1 attaches to the dimer through activation by photons in the wavelength range of 320 – 450 nm, thereby removing the dimers and reversing the damage [4].

Ionizing radiation (IR) can be the cause of serval different DNA lesions. The damage can be contributed to lack of a specific target. The most common reaction associated in IR is its interaction with water. It can destabilize H2O molecules creating hydroxyl radicals, one of the ROS mentioned earlier. These hydroxyl radicals can attack the phosphate backbone of DNA and cause single stranded breaks [4]. This gives the hydroxyl radicals an opportunity to oxidize specific bases since they are no longer shielded by the double helix conformation. The most significant base to be affected is guanine, creating 8-hydroxyguanine, which can easily pair with both adenine and cytosine bases and can be recognized by varying polymerases for replication and transcription [4]. Single- stranded breaks (SSB) are repaired by means of DNA ligase. IR and UV-C can also lead to double- stranded breaks (DSB), which are more difficult to repair due to random ligation of either end by DNA ligases, causing possible “illegitimate recombination”, resulting in nucleotide sequence alterations (mutation) [4].

Alkylation damage is the spontaneous methylation of bases that can cause mismatched pairing. Most cells have extensive pathways to manage DNA damage with the most common cause being endogenous methylations agents [4][5]. The nuclear excision repair (NER), base excision repair (BER), and mismatch repair (MMR) pathways are some of the key mechanisms for repairing alkylation damage [6][7]. The alkylating agent, ethylmethane sulfonate (EMS), is commonly used in genetics research to induce point mutations in plants [8]. Most pairing caused by EMS mutation will either not change pairing preference, but some do the exact opposite and will not allow pairing with any base, usually

2 leading to a halt in replication, transcription and translation. On occasion it will create the product, O6-alkylguanine, which will bind freely to adenine. After a successful replication, this will cause a guanine to adenine point mutation [4] [8].

In the present study, the genomutagenic compound bleomycin (BLM) was used to induce DNA damage. This compound is used as a chemotherapy drug to battle lymphomas,

[9] neck, head and testicular cancers . When bleomycin interacts with O2 and a metal (usually

Fe(II) or Cu(I)) it is converted into its active form, active bleomycin (ABLM). ABLM can bind to DNA and cleave G-C bonds, causing either single or double stranded breaks. This compound is a good substitute to simulate ionizing radiation due to the similar symptoms and pathways for DNA repair [10][11].

Damage Suppressor Protein (Dsup) from Tardigrade

The tardigrade, also known as water bear, are considered by many to be the world’s most hardy animal. Measuring in at 1 mm in length at their largest, these tiny creatures can withstand a plethora of extreme environments and are found in almost all aquatic environments all over the earth. When they are introduced to a dry environment, they enter an anhydrobiotic state. In a desiccated state, they can withstand extreme temperatures, potentially toxic chemicals, ionizing radiation, UV radiation and have even been known to survive in the vacuum of space [12]. This animal is now being used as a model organism to study the rigorous effects of space travel due to the complex nature of this microscopic organism [13].

This organism’s ability to defend itself from these extreme environments caught the attention of a research group from Japan. A team led by Takekazu Kunieda isolated unique chromatin-associated proteins found in an extremotolerant species of tardigrade

3 called Ramazzotius varieornatus. They expressed these proteins in Drosophila cell cultures using a GFP tag and discovered a single protein called Damage Suppressor Protein (Dsup) which showed nuclear localization. The Dsup gene was introduced and expressed in the

HEK293 human cell lines [14][15]. Transformed and untransformed cell lines were X-ray irradiated followed by analysis of DNA damage using an alkaline comet assay. The assay places cells in an alkaline solution to denature double-stranded DNA followed by single cell electrophoresis to measure single stranded breaks in the nuclear genome. The longer the “tail” the more damage done to the DNA. Tails found in Dsup expressing cells were reduced by 50% compared to the untransformed cells [14][15].

Samples were then irradiated and subjected to a γ-H2AX immunofluorescent assay.

When double stranded breaks occur, H2AX histones become phosphorylated and can be visualized by immunofluorescence. Fluorescent foci were counted and the Dsup expressing cell lines showed a 40% reduction in observed foci. Under treatments of high X-ray radiation, cells were also tested for viability. Untransformed cells showed abnormal round morphology while the Dsup expressing cells showed normal morphology. Dsup cells also showed a slightly faster proliferation rate compared to untransformed cell lines. This evidence shows that Dsup transformed cell lines had a greater viability compared to their untransformed counter parts when irradiated under X-ray [14][15].

Single Cell Gel Electrophoresis (Alkaline Comet Assay)

The alkaline comet assay was first published in 1984 by Ostling and Johanson [16].

It has become a widely used technology to access damage by single stranded and double stranded breaks to nuclear genomes in eukaryotic cells [17]. New protocols have even

4 studied NER and BER repair pathways by allowing cells to repair before cell lysis is conducted [18].

Briefly, isolated single cells are placed into agarose gel and lysed. This solidifies nuclei in the gel where the intact cell once previously. Samples are then washed and subjected to gel electrophoresis to separate smaller fragments of DNA from the larger undamaged sections of the genome. The gel is then dyed with a fluorescent DNA binding chemical and viewed under a fluorescent microscope. This shows a visible signal as a

“head” where the nucleus is located and a “tail” of damaged DNA running from the head.

The longer the tail the more breaks that are present in the nucleus. For our experiments we have modified multiple protocols in order to create a lysis/electrophoresis buffer combination most suited to our needs working with tobacco [19][20].

Dsup expression in plants

Based on the observation of the effects of Dsup on human cell lines, we wondered what effect the tardigrade Dsup protein might have on plants growth and resistance to DNA mutation and other stresses, as well as its effect on expression of the endogenous, DNA damage response and repair related genes. We hypothesize that the presence of the Dsup protein may lead to reduction of DNA damage caused by genomutagenic sources in plants expressing this protein. We have used Agrobacterium mediated transformation method to create tobacco plants that express Dsup. This study was conducted by means of molecular and plant growth analysis. We have also developed our own optimized protocol for an alkaline comet assay to assess the viability of our Dsup lines in comparison to control lines.

5 Methods and Materials

Generation of Transgenic Plants

The model plant tobacco, Nicotiana tabacum, was chosen due to its quick maturation, easy care and successful viability for transformation. Transgenic lines were generated via Agrobacterium-mediated transformation described in [21 – 24]. The modified

Agrobacterium tumor inducing (Ti) plasmid used was designed by Dr. Zhang by use of the nucleotide sequence published on NCBI. The coding sequence of the gene of interest,

Dsup, was codon optimized for the tobacco genome and was fused to the CaMV35 super promoter. NPTII gene (kanamycin resistance gene) is promoted by the NOS promoter.

Both genes are terminated by the NOS terminator. The plasmid construction is shown in

Figure 1. Transgenic lines were generated from a previous transgenic tobacco line RM1, which was modified to express the GUS protein. This was done in the hope of being able to quickly identify mutations in our lines by histochemical staining using X-Gluc. NPTII was chosen as a selection marker due to RM1’s loss of NPTII function in the previous transformation.

6

Figure 1: Engineered Ti plasmid containing Dsup and NPTII genes driven by NOS and

CaMV-35S promoters.

Leaf disks were infected with the pNtDsup Agrobacterium culture and were grown on selection RMOP media containing kanamycin and timentin (used to eliminate any remaining Agrobacterium) to allow for development of transgenic shoots. Shoots were then placed into rooting medium containing kanamycin and timentin to continue selection. Once the plants were large enough, they were transplanted into soil pots to grow to maturity (T0 generation), until seed pods were collected. These seeds were our first transgenic generation known as T1.

The T1 seeds were surface sterilized in 10% bleach solution containing Tween 20 and placed into seed germination media containing kanamycin for selection. Soon after seedling sprouting, the segregation ratio (resistant vs sensitive to kanamycin) was scored to infer copy number of the Dsup transgene. The kanamycin resistant seedlings were placed into rooting boxes. When large enough, they were planted in soil to continue mature and flower. The leaf samples were collected for DNA or RNA analysis (see below).

Homozygous seeds (T2) were then collected, designated as DsupR, and used for further study.

7 Growth Analysis

The samples we chose for this analysis included RM1 and R5-3. We also chose two different genomutagenic chemicals in different concentrations as treatments added directly into seed germination media. Treatments included 1 mM EMS, 3 mM EMS, 1 µg/ml bleomycin, 3 µg/ml bleomycin, and a control (no genomutagen). Approximately 30 seeds from each of our samples of interest were germinated in media containing one of the treatments listed. Seeds were left to germinate and grow for two weeks then collected.

Measurements were taken for fresh weight and root growth. Results were analyzed for statistical significance using paired t-test in R statistics software.

Histochemical Staining analysis

The experiment was conducted by means of regenerating leaf disk tissue on media containing EMS varying concentrations of EMS at 0 mM, 1 mM, and 5 mM. RM1 was tested first to determine if there was a notable change in GUS activity in the leaf tissues. A separate plate was also made to treat seedlings with UV-C radiation delivered by a UV lamp. T0 generation was tested next using samples RM1, R1, and R2. Seedlings from R1 and RM1 were also germinated on media containing the same concentrations of EMS.

Leaves and roots were collected from these samples and compared. Once T1 generation was generated we repeated the experiment with sample R2-2, R2-3, R4-3 and R5-3 collecting GUS stain samples at 2 weeks and 4 weeks.

DNA and RNA extraction, cDNA synthesis, and qPCR analysis

During the growth of each generation, Quantitative PCR (qPCR) and Reverse

Transcriptase qPCR (RT-qPCR) analysis was preformed to confirm that our gene of interest was present in the genome and being transcribed into mRNA.

8 DNA was extracted from ~100 mg fresh leaf using an Omega E.Z.N.A. Plant DNA

Kit. DNA isolates were analyzed using a nanodrop spectrophotometer to calculate DNA concentration. DNA solutions were then diluted to 2 ng/µl to preform qPCR analysis.

RNA was extracted from leaves using a Geneaid Total RNA Mini Kit (Plant), with addition of DNase to remove the residue genomic DNA. RNA isolates were analyzed using a nanodrop spectrophotometer to calculate RNA concentration. RNA samples were reverse transcribed to generate cDNA using a Lunascript RT Supermix Kit. The cDNA was diluted to 1.5 ng/µl to be used in RT-qPCR.

qPCR and RT-qPCR were performed on an Applied Biosystems StepOnePlus Real-

Time PCR System. Statistical analysis was performed by the ΔΔCt method to conclude the relative gene dosage of DNA or relative expression of RNA. Reference gene EFα was used to compare to our target gene.

Analysis of DNA Damage Targets through RT-qPCR (Damage Assay)

Experiments for this assay required the same treatment setup described in the growth analysis. Seedlings were grown for two weeks. Plantlets were collected, and RNA was extracted and put through a reverse transcriptase treatment to create a cDNA library as described previously. For X-ray and UV-C treatments, 20 – 30 seeds of RM1 and R5-3 lines were germinated on sterile seed germination media. Two plates were prepared for both treatment and control (untreated) samples. After 2 weeks, samples were treated by

UV-C exposure or X-ray exposure. UV-C exposure was conducted by placing plates into a box and placing a UV-C (~ 240 nm) lamp 20 cm from the plate for 30 minutes. This calculates to an exposure dosage of 5.59x10-17 J. X-ray exposure was preformed using a

Bruker Skyscan1173 micro-CT Scanner at 80 kilovolts and 44 microamps at a distance of

9 188 mm for ~2 minutes. We allowed 45 minutes before collection to allow molecular response to the damage. Whole seedling samples were then collected from both the treatment and control groups, then frozen in a -80° C freezer until RNA extraction. qPCR was performed on the samples using target genes reported to be involved in DNA damage detection and repair.

Single Cell Gel Electrophoresis (Alkaline Comet Assay)

Method for this assay are modified protocols initially developed by Olive and

Georgieva [19][20]. Different elements of their protocols were used to supplement our needs and means. Samples were prepared as mentioned in the Damage Assay section for X-ray and UV-C treatments, apart from seedlings being collected immediately after treatment to ensure the plant does not undergo repair to the nuclear genome.

Slides where etched around the edges using a tungsten carbide pen to increase the adherence of agar. Following cleaning with detergent and then with ethanol, a layer of 1% agar was placed onto a glass slide and chilled for 10 minutes. Nuclei were isolated by cutting entire seedlings with a razor blade in ice cold nuclei isolation buffer. Samples were then drip filtered using a 20µm filter. A sample (40µl) of nuclei suspension was mixed with

60µl of 1% low melt agar, and the mixture was placed on top of the layered slides. The slide was again chilled to allow for solidification of low melt agar.

Slides were placed into ice cold lysis buffer (1.2 M NaCl, 100 mM Na2EDTA, 50 mM Tris, 0.1% N-Lauroylsarcosine sodium salt, 1% Triton X-100, 10% DMSO, pH to ~10 using NaOH) for one hour to lyse the nuclear membrane and any remaining excess cellular components. Samples were then washed for 20 minutes in cold electrophoresis buffer (30 mM NaOH, 500 mM Na2EDTA) before being placed into a gel tank with fresh buffer for

10 electrophoresis for 25 minutes at 0.6 volts/cm (9 volts in our 15 cm tank). Slides were then neutralized by washing in deionized water for 10 minutes and then dyed using 10 µg/ml acridine orange solution. After 20 minutes of dye samples were then de-stained in deionized water.

When bound to DNA this dye will emit green (525 nm) fluorescence when excited by a max of 500 nm light (cyan). Imaging of slides was performed using a Nikon Eclipse

Ts2R epi-fluorescent microscope with a 470 nm excitation filter. Images were captured using the NIS Elements software and analyzed using CASPLab comet assay software.

Results were analyzed for statistical significance using paired t-test in R statistics software.

11 Results

Bioinformatics Analysis

Initial searching for the Dsup gene was done with NCBI genome database. We found the nucleotide sequence for the gene with given accession number “LC050827”. The

Dsup protein is a 445 amino acid sequence, containing 3 functional regions, the N- terminus, α-helical region, and the C-terminus (Figure 2). Based on the research, the C- terminus seems to be the region associated with nuclear localization [14][15]. Initial searches on the Swiss image depository came up with two separate images based on the amino acid sequence. Figure 3 shows results from entering the protein sequence from UniprotKB’s amino acid sequence “P0DOW4” into the database. The structure of Dsup is thus far unknown and the image was provided by I-TASSER protein modeling software.

Figure 2: Amino acid sequence for Dsup protein based off ascension “P0DOW4”.

Highlighted in yellow is the N-terminus, underlined is the α-helical region, and

highlighted in green is the C-terminus. Boxed in Red is a possible nuclear localization

signal (NLS)

12

Figure 3:I-TASSER Predicted 3D structure of the Dsup Protein

I continued by searching the database for possible homologs or orthologs to confirm the uniqueness of this protein. pBlast searches for all three regions mentioned above, as well as for 3 regions showed composition bias. The results showed that the C-terminus only comparison was to the Dsup gene while the other two regions had single results shown in Figure 4. The results would seem unreliable due to the low identity percentage, lack of any conserved domains, and high error values (E-value) shown on the report. In the pBlast for the specific amino acid sequences, we only found the Dsup protein. This information helped to confirm that this was a truly unique protein not found in any other organisms.

Figure 4: pBlast results for α-helical region of the Dsup protein

13 Genotyping and Expression of Dsup Transgenic Plants

After the testing of multiple lines, we chose samples R5-3 as our best candidate for further mutagenic testing due to the large relative copy number (Figures 5 and 6) and high rate of mRNA expression of the Dsup gene (Figure 7).

Relative Gene Dosage Of Dsup in T0 Plants 2.5

2

1.5

1

0.5

0 Rm1 R1 R2 R3 R4 R5 R6

Figure 5: Relative gene dosage of Dsup gene for T0 plants based on reference gene EFα.

Relative Expression of Dsup mRNA in T0 plants 2.5

2

1.5

1

0.5

0 Rm1 R1 R2 R3 R4 R5 R6

Figure 6: Relative expression of Dsup mRNA in T0 plants based on reference gene EFα.

14 Relative Gene Dosage Of Dsup in T1 plants 8 7 6 5 4 3 2 1 0 R2-T0 R4-T0 R5-T0 R2-1 R2-2 R2-3 R4-1 R4-2 R4-3 R5-1 R5-2 R5-3

Figure 7: Relative gene dosage of Dsup gene for T1 plants based on reference gene EFα.

Histochemical Staining analysis

The RM1 tobacco line was chosen because of its transgenic addition of the GUS protein was available, which should be a good marker to help identify damage done to the genome. Any GUS activity that would have been inhibited by mutagenic sources would result in a reduction of blue precipitate in the plant seedlings, leaf disks, and roots. This seemed to be the case in preliminary testing with RM1 and T0 generation (Figure 8), but no quantifiable data was obtained. In later tests, including the Dsup transformed plants, we noticed unreliable patchy expression of the GUS protein making it nearly impossible to determine if the plants had a reduction of GUS activity by eye.

15

Figure 8: GUS stain analysis of T0 tissue regeneration on EMS media.

Growth Assay

Figures 9 to 12 below depict the results shown for the growth assay performed under multiple concentrations of EMS. 0 mM (control), 1 mM and 3 mM concentrations were tested. No change was seen in control samples while RM1 showed a reduction in fresh weight and root length of the plant in 1 mM EMS. In the same concentration, R5-3 seemed to be nearly unaffected by the mutagenic chemical. This also depicts that the effects of damage reduction are limited with 3 mM EMS causing no change between treated control and Dsup sample lines.

16 Average Fresh Weight of EMS treated Samples 25 20 * 15 10 *

5 Fresh Fresh Wieght (mg) 0 RM1 R5-3

0 mM 1 mM 3 mM

Figure 9: Average fresh weight of EMS treated samples. *Significant difference found

between treatment samples with p-value < 0.05. p-value = 0.0001262

Below are the results of the paired t-test performed for BLM treated samples of

RM1 and R5-3 to show significant difference in the average fresh weight of samples

(Figure 9). As shown, the p-value is less than 0.05 confirming significant results.

Paired t-test

data: data$RM and data$R5 - 3

t = -4.7939, df = 19, p-value = 0.0001262

alternative hypothesis: true difference in means is not equal to 0

95 percent confidence interval:

-7.843824 -3.076176

sample estimates:

mean of the differences

-5.46

17 Average Root Length (mm) of EMS treated Samples 20

15 *

10 *

5

Root Length(mm) 0 RM1 R5-3

0 mM 1 mM 3 mM

Figure 10: Average root length of EMS treated samples. *Significant difference found

between treatment samples with p-value > 0.05. p-value = 7.465e-05.

Below are the results of the paired t-test performed for BLM treated samples of

RM1 and R5-3 to show significant difference in the average root length of samples (Figure

10). As shown, the p-value is less than 0.05 confirming significant results.

Paired t-test

data: data$RM and data$R5 - 3

t = -5.5262, df = 14, p-value = 7.465e-05

alternative hypothesis: true difference in means is not equal to 0

95 percent confidence interval:

-9.448407 -4.164927

sample estimates:

mean of the differences

-6.806667

18

Figure 11: Growth assay plates for RM1 under 0 mM, 1 mM and 3 mM EMS treatment.

Figure 12: Growth assay plates for R5-3 under 0 mM, 1 mM and 3 mM EMS treatment.

Analysis of DNA Damage Targets by RT-qPCR (Damage Assay)

In order to examine whether Dsup expression affected the expression of the endogenous genes in plants under different treatments, RT-qPCR was performed to analyze genes involving DNA damage response and repair. Eighteen different target genes are listed and shown in Table 1. It shows the difference in gene expression of these targets between RM1 lines and Dsup line R5-3. Compared to the control plant RM1, either upregulated, downregulated or no change in gene expression in the Dsup-expressing plant

Dsup R5-3, as well as annotation for gene ontology are shown in Table 1. 19 Target Gene Ontology BLM UV-C X-ray Treatment Treatment Treatment ATM DNA Damage signaling; SSB ↓ ≌ ↓

ATR DNA Damage signaling; DSB ↑ ↑ ↑

BT3/4 Telomerase transcription regulation ≌ ≌ ≌

CASP1/2 Apoptosis mediation ≌ ↓ ≌

CASP3/4 Apoptosis mediation ↓ ≌ ↑

CASP5/6 Apoptosis mediation ↓ ↓ ↑

Cen2 Signaling Complex; Involved in NER ↓ ≌ ≌

DDB Damaged DNA binding ≌ ≌ ↑

DDM Helicase activity, ATP binding ↑ ↑ ≌

ERCC DNA repair helicase ≌ ↓ ↑

Ku70 DNA damage helicase ≌ ≌ ≌

Parp1 DNA damage signaling ↓ ↑ ↑

Parp2 DNA damage signaling ↓ ↑ ↑

RAD17 Cell cycle checkpoint ≌ ≌ ≌

RAD23 DNA binding; Involved in NER ≌ ↑ ↑

RAD51 Double stranded break repair ≌ ↑ ↑

RAD513 Double stranded break repair ≌ ≌ ≌

TGA DNA damage transcription factor ↓ ↓ ↑

Table 1: Target genes tested for expression level changes in Dsup vs Control plants

treated with genomutagens. ↓ showed downregulation, ↑ showed upregulation, and ≌

showed no change in gene expression.

Selective RT-qPCR results in detail for BLM treated samples are shown in Figures

13 to 16, illustrating examples of upregulated, downregulated and no change in target RNA expression.

20 ATR 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 RM R5-3 RM+B R5-3+B

Figure 13: Relative gene expression of ATR. Example of upregulation between treated

samples.

TGA 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 RM R5-3 RM + B R5-3 + B

Figure 14: Relative gene expression of TGA. Example of downregulation between treated

samples.

21 CASP5/6 7 6 5 4 3 2 1 0 RM R5-3 RM + B R5-3 + B

Figure 15:Relative gene expression of CASP5/6. Example of downregulation to normal

levels between treated samples.

Ku70 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 RM R5-3 RM + B R5-3 + B

Figure 16: Relative gene expression of Ku70. Example of no change in regulation

between treated samples.

Selective RT-qPCR results in detail for UV-C treated samples are shown in Figures

17 to 20. This shows examples of upregulated, downregulated and no change in target RNA expression.

22 ERCC 4 3.5 3 2.5 2 1.5 1 0.5 0 RM R5-3 RM+UV R5-3+UV

Figure 17: Relative gene expression of ERCC. Example of downregulation to normal

levels between treated samples.

TGA 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 RM R5 RM+UV R5-3+UV

Figure 18: Relative gene expression of TGA. Example of downregulation to normal levels

between treated samples.

23 RAD51 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 RM R5-3 RM+UV R5-3+UV

Figure 19: Relative gene expression of RAD51. Example of upregulation between treated

samples.

DDB 3.5 3 2.5 2 1.5 1 0.5 0 RM R5-3 RM+UV R5-3+UV

Figure 20: Relative gene expression of DDB. Example of no change in regulation

between treated samples.

Selective RT-qPCR results for X-ray treated samples are shown in Figures 21 to

24. This shows examples of upregulated, downregulated and no change in target RNA expression.

24 ATM 1.2

1

0.8

0.6

0.4

0.2

0 RM R5-3 RM Xray R5-3 Xray

Figure 21: Relative gene expression of ATM. Example of downregulation to normal

levels between treated samples.

ATR 2.5

2

1.5

1

0.5

0 RM R5-3 RM Xray R5-3 Xray

Figure 22: Relative gene expression of ATR. Example of upregulation between treated

samples.

25 TGA1/2 2.5

2

1.5

1

0.5

0 RM R5 RM+Xray R5-3+Xray

Figure 23: Relative gene expression of TGA. Example of upregulation between treated

samples.

CEN2 9 8 7 6 5 4 3 2 1 0 RM R5-3 RM Xray R5-3 Xray

Figure 24: Relative gene expression of CEN2. Example of no change in regulation

between treated samples.

Overall, our gene expression analysis indicates that expression of the tardigrade

Dsup gene in tobacco plants altered the patterns of some endogenous gene expression such as ATM, ATR, TGA, PARP1, PARP2, CASP5/6. All these genes are invovled in DNA damage response and repair pathways when the plants are under stress treatments.

26 Single Cell Gel Electrophoresis (Alkaline Comet Assay)

Figures 25 to 28 depict the alkaline comet assay preformed on control and UV-C treated samples. RM1 samples showed a reduction in head size and an increase in tail size/movement (Figure 25). R5-3 showed an opposite effect when treated with UV-C

(Figure 26). The size of the head increased dramatically, and a tail was greatly reduced

(Figure 27). Similar results were shown from X-ray treated samples (Figure 28).

Figure 25: Alkaline comet assay images for RM1 samples.

27

Figure 26: Alkaline comet assay images for R5-3 samples.

Tail Movement for UV Treatment 140 120 * 100 80 60 40 20 * 0 RM R5-3 RM+UV R5-3+UV

Figure 27: Average tail movement for UV-C treated samples. *Significant difference

found between treatment samples with p-value < 0.05. p-value = 2.2e-16

28 Below are the results of the paired t-test performed for UV treated samples of RM1 and R5-3 to show significant difference in the average tail movement of comets (Figure

27). As shown, the p-value is less than 0.05 confirming significant results.

Paired t-test

data: data$RM and data$R5 - 3

t = 14.493, df = 74, p-value < 2.2e-16

alternative hypothesis: true difference in means is not equal to 0

95 percent confidence interval:

85.19397 112.35327

sample estimates:

mean of the differences

98.77362

Tail Movement for X-ray Treatment 12 * 10

8

6

4 *

2

0 RM R5 RM+Xray R5-3+Xray

Figure 28: Average tail movement for X-ray treated samples. *Significant difference

found between treatment samples with p-value < 0.05. p-value = 9.221e-09

29 Below are the results of the paired t-test performed for X-ray treated samples of

RM1 and R5-3 to show significant difference in the average tail movement of comets

(Figure 28). As shown, the p-value is less than 0.05 confirming significant results.

Paired t-test

data: data$RM and data$R5 - 3

t = 6.3411, df = 89, p-value = 9.221e-09

alternative hypothesis: true difference in means is not equal to 0

95 percent confidence interval:

5.078632 9.713855

sample estimates:

mean of the differences

7.396244

These results suggest that he Dsup-expressing cells exhibited much less genome damage under either UC-C or X-ray than the Dsup-lacking control cells.

30 Discussion

With data shown from qPCR and RT-qPCR for the Dsup gene we can confirm that our plants lines were transformed during Agrobacterium transfection. We had multiple lines transformed but due to the high gene dosage levels found in genomic DNA extractions and high levels of mRNA shown from RNA extraction, the R5-3 line was chosen to continue testing and analysis.

Through the growth analysis we saw a much higher survivability in the R5-3 lines over RM when germinated in media containing 1 mM EMS. R5-3 samples showed almost normal growth in this media while RM1 showed reduced and abnormal growth. This observation was supported by fresh weight and root growth measurement data shown on the graphs in figures 9 and 10. The effects of the presence of the Dsup protein seem to be limited due to a lack of change in health when germinated in 3 mM EMS growth media.

Fresh weight and root growth data also supports this limiting concentration of the mutagenic chemical.

BLM was also a candidate for this analysis but the results from the growth analysis were deemed unreliable. This may be due to a phenomenon observed while preparing the growth media where precipitate was formed while adding BLM. This may have elevated or lowered concentrations of the chemical in treatment plates. Another biological repeat of this experiment will need to be conducted to obtain reliable data for future study.

Another method of analysis that we attempted was a histochemical staining analysis. To perform this assay, we did our initial transformations with RM1, which was

31 pre-transformed with the GUS gene. This way we could monitor visually if GUS activity was inhibited. However, expression patterns for GUS were unreliable in both RM1 lines and R5-3. It was not visually discernible if GUS expression was interrupted or retained.

Further experiments will be planned to continue the use and function of this molecular tool that is within our Dsup-R lines. We will make future attempts at a quantitative GUS enzyme activity assay to obtain data that can show possible changes.

For our RT-qPCR analysis for DNA damage and repair targets we had chosen 18 gene candidates for testing based on their involvement in DNA damage detection and repair pathways. This analysis was designed to determine changes in gene expression due to the presence of the Dsup protein. Two sample lines and three separate treatments were tested for each target. BLM treatments showed 2 genes upregulated, 7 genes downregulated and

9 genes with no changes in expression. UV-C treatments showed 6 genes upregulated, 4 genes downregulated and 8 genes with no changes in expression. X-ray treatments showed

10 genes upregulated, 1 gene downregulated and 7 genes with no changes in expression.

Table 1 in the results section outlines each target, its general function, and the change in mRNA expression exhibited from RM1 lines to lines containing the Dsup protein (R5-3).

Gene ATM, which is involved in single stranded break (SSB) signaling [25], showed a downregulated expression in comparison from R5-3 to RM1 in BLM and X-ray treatment. This could give us a hint that the Dsup protein may play a role in protection from SSB causing less signaling to occur. While on the other hand, the gene ATR, which is involved in DSB signaling showed upregulation for all treatment conditions [25]. This shows that there may be a link to the Dsup protein and an enhanced signaling for DSB’s,

32 which would be significant due to DSB being the most common damage caused by all three sources.

Other genes involved with DNA damage signaling were also heavily affected by the presence of Dsup. Parp1 and Parp2 showed upregulation when treated with ionizing radiation and UV-C, which both cause direct damage to DNA [25][26]. While under mutagenic chemical condition with BLM showed downregulation and the possibility of

BLM reacting with Dsup to inhibit damage from this source requiring less signal to induce repair. The upregulation of signaling also correlates with targets involved with damage repair and the NER pathway. RAD23 (DBS repair) and RAD51(NER pathway repair), both exhibited upregulation and possible enhanced repair process after treatment with X-ray and

UV-C, while neither of their gene expression was changed under BLM conditions [25].

For genes involved with DNA repair mechanisms, three targets (DDB, DDM, and

ERCC) were chosen for their DNA repair helicase activity showed up no noticeable trend in either direction [25]. DDB showed upregulation in only the X-ray treatment, DDM showed upregulation for BLM and UV-C, and ERCC showed upregulation for UV-C treatments, and downregulation for X-ray treatments.

We also looked at three CASP genes to see if Dsup had any affect over apoptosis control. Our results showed no clear trend. UV-C and BLM treatments showed down regulation of two of our targets, while X-ray showed upregulation with two of the targets.

This downregulation of these targets may mean that the cells are not under as much stress to preform apoptosis. Although no trend was seen with helicase or CASP genes, the transcription factor TGA, known for its role for controlling DNA damage proteins showed downregulation for BLM and UV-C and upregulation for X-ray treatments [27]. Many more

33 genes are found in the different pathways for damage detection and repair. Future research in this project should include a transcriptome analysis to compare a much larger set of gene targets. Due to time constraints, it would have not been possible to complete this analysis during this current project.

Our comet assay also showed evidence for the Dsup protein’s proposed DNA protective properties. In both the X-ray and UV-C treatments R5-3 cells showed a reduction in tail movement. This confers the idea that less DNA damage leads to slower movement of the DNA “tail”. After treatment, R5-3 lines showed an interesting trend. The

“head” of the comet was enlarged compared to all other samples. This enlargement may be a response to the damage caused by the mutagenic sources. The presence of Dsup proteins may be protecting DNA from fragmentation, resulting in less movement, and keeping relative directions of sense and anti-sense strand in order to avoid recombination. Little is known about the actual mechanisms of the Dsup protein other than its nuclear localization, so more research on its mechanism and DNA binding properties will have to be conducted to substantiate this claim.

With the improved growth shown in EMS media and results shown from the RT- qPCR and comet analyses, we feel that our hypothesis is proven and the Dsup protein is showing improved survival of plants to genomutagenic sources. While the study seemed successful, some aspects of it needed to be improved. As mentioned, issues with the solubility of the chemical mutagens left data that was unusable as evidence. While we did discover that pre-diluting the solution helped us avoid precipitation, we will still have to repeat these experiments to further our understanding as to how EMS will affect gene expression of the Dusp plants. We will also need to eventually conduct multiple biological

34 repeats and testing with other lines transformed by wild type tobacco to ensure that results can be replicated and to have a functioning product lacking the additional GUS expression found in the Dsup-R lines.

As we continue, more study will have to be conducted to fully understand the pathways and mechanisms affected by the presence of this protein but hopefully this work has laid the groundwork to further investigation and possible use in environments that would be considered harsh to an organism genome, such as future colonization of space or in areas effected by radiation such as Fukushima, Japan [28].

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