Transcriptomic Analysis of the Heat-Stress Response in Boechera Depauperata and Arabidopsis Reveals a Distinct and Unusual Heat-Stress Response in Boechera

Botany

Transcriptomic analysis of the heat-stress response in Boechera depauperata and Arabidopsis reveals a distinct and unusual heat-stress response in Boechera.

Journal: Botany

Manuscript ID cjb-2020-0014.R2

Manuscript Type: Article

Date Submitted by the 21-May-2020 Author:

Complete List of Authors: Pierce, Ian; San Diego State University, Biology Halter, Gillian; San Diego State University, Biology Waters, ElizabethDraft R. ; San Diego State University, Biology Keyword: Arabidopsis, heat-stress, transcriptomics, RNA-seq

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

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Title: Transcriptomic analysis of the heat stress response in Boechera depauperata and

Arabidopsis reveals a distinct and unusual heat stress response in Boechera.

Authors: Ian Piercea, Gillian Haltera, and Elizabeth R. Watersa, *

Affiliations:

a Department of Biology, San Diego State University, San Diego, CA. 92182

* Corresponding author: [email protected] Draft

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Abstract: Global surface temperatures are expected to rise throughout the 21st century

and will negatively impact plant growth and reproduction. Thus, it is imperative that we

deepen our understanding of plant thermotolerance. The examination of native plant

species that have evolved tolerance to high temperatures can provide crucial

information on how plants can adapt to climate change. Boechera (Brassicaceae), a

large genus that is native to North America, is highly thermotolerant and can maintain

photosynthetic activity at high temperatures. Here we report results of transcriptomic

studies that seek to reveal possible thermotolerance mechanisms in B. depauperata.

Analysis of RNA-seq datasets from heat stressed B. depauperata and Arabidopsis

thaliana plants identified significant differences in how each of these species responds

to identical heat stress conditions. TheDraft most highly upregulated heat-stress genes in A. thaliana includes the well-characterized heat-shock genes. In contrast, the Boechera heat stress response is composed of: novel genes that lack orthologs in other genomes, genes coding for proteins of uncharacterized function, and genes coding for proteins associated with the unfolded protein and ER stress responses. In addition, genes that are protective of photosynthetic capacity are also differentially upregulated in B. depauperata.

Key words: Arabidopsis, Boechera, heat stress, transcriptomics, RNA-seq, orphan genes.

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Introduction

Plants are sessile organisms, and this means that wherever a seed germinates

the growing plant must survive to produce the next generation or that lineage becomes

a dead-end. Thus, tolerance to stress is a crucial aspect of plant success. As our

climate changes the severity and duration of heatwaves are expected to increase

(Stocker et al. 2013). Soon, intensifying heat stress will have a greater impact on plant

growth; and this stress will challenge the extent of plant adaptation and tolerance to

heat-stress. This, in turn, puts both crop productivity and the survival of native plant

species at risk. Thermotolerance is a complex and crucial trait for plant growth and

reproduction and its importance in plants has motivated a large number of studies in

crop and model plant species. The best-studiedDraft model species is Arabidopsis thaliana.

Much of what we know about thermotolerance, the heat shock response, and the heat

shock proteins in plants is based on studies in Arabidopsis. The enormous body of

literature on Arabidopsis and heat stress is impossible to cite here. However, there have

been a few seminal papers that provide a mechanistic understanding of

thermotolerance in Arabidopsis, in particular, and in all plants, in general (Larkindale et

al. 2005; Kotak et al. 2007; Larkindale and Vierling 2008; Saidi et al. 2011; Mittler et al.

2012). What we know about heat stress in Arabidopsis (and for most plants as well) is

that exposure to a short-term (few hours) of moderate heat stress at 38°C is not lethal

and that this stress induces the expression of the heat shock proteins (HSPs) (Vierling

1991; Waters et al. 1996; Queitsch et al. 2000; Kotak et al. 2007). The HSPs are

molecular chaperones and as such, they protect cells from heat-induced damage (

Hendrick and Hartl 1993; Haslbeck and Buchner 2002; Haslbeck et al. 2005; Hilton et

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al. 2013; Kim et al. 2013). However, there is an upper limit of heat stress above which

(for most plants including Arabidopsis this is above 44° or 45°C) even the HSPs cannot provide protection and cell death occurs (Larkindale et al. 2005; Kotak et al. 2007).

While it is recognized that the heat shock proteins are a crucial component of thermotolerance and are one the best studied responses to stress, it is now widely understood that the heat shock proteins (HSPs) comprise only one component of thermotolerance (Hanson et al. 1999; Sharkey 2000; Larkindale et al. 2005; Kotak et al.

2007; Larkindale and Vierling 2008; Sharkey and Zhang 2010) see also (Mittler et al.

2012; Ohama et al. 2017; Scheepens et al. 2018). Most of the thermotolerance studies are conducted using standard thermotolerance methodologies as described in (Yeh et al. 2012) and use Arabidopsis as theirDraft study species.

Recently, the importance of understanding thermotolerance for plant conservation has been acknowledged and this has stimulated thermotolerance studies in a wider range of plants (French et al. 2017). Further, it is clear that detailed knowledge of thermotolerance in naturally thermotolerant species will greatly increase our understanding of the physiological and biochemical mechanisms that underlie this important trait. The value of studies in Arabidopsis is that they benefit from a well- curated genome and decades of past studies that provide an important foundation for molecular and physiological studies. However, by only focusing on just one species we risk considerable bias in our understanding of the possible range of thermotolerance levels and in the stress response itself. In our previous studies (Gallas and Waters

2015; Halter et al. 2017; Kannan et al. 2018) we have examined the thermotolerance and stress responses of a group of native California plants in the Boechera genus

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(Brassicaceae). Boechera is a large genus over 100 species that are native to North

America (Al-Shehbaz 2003; Alexander et al. 2013). The Boechera are found from cool

moist coastal environments to high elevation mountains, to hot and dry deserts (Gallas

and Waters 2015; Halter et al 2017). A phylogenetic analysis of the Brassicaceae

family identified three major lineages (I, II, III) (Franzke et al. 2011). Both the

Arabidopsis and Boechera genera are members of the well-supported Lineage I within

the Brassicaceae. Thus, studies of the Boechera provide both the value of being related

to the best-studied model plant and with it the massive body of knowledge of the

Arabidopsis stress responses, as well as the value of examining plants that have

adapted to varied and extreme natural environments. In our studies, we have found

considerable diversity in levels of abioticDraft stress tolerance among the Boechera species

(Gallas and Waters 2015; Halter et al. 2017). Notably, we found that all the Boechera

species studied were more thermotolerant than Arabidopsis and that among the

Boechera species examined, Boechera depauperata is the most thermotolerant.

Further, our studies utilizing qPCR of the well-studied heat shock genes have shown

that the high levels of thermotolerance in the Boechera are not correlated with high

levels of HSP gene expression (Gallas and Waters 2015; Halter et al 2017). In addition,

we found that when compared to A. thaliana, B. depauperata has a much higher ability

to protect photosynthetic capacity during heat stress (Halter et al 2017).

In order to fully understand B. depauperata’ high level of thermotolerance, we

have analyzed the heat stress transcriptomes of Boechera depauperata and the related

model plant species Arabidopsis thaliana. Here we report significant differences in the

heat stress response between these two related species. Most of the most highly

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expressed genes in A. thaliana are the well-known heat shock genes. However, the most highly expressed genes in B. depauperata included novel genes not found in A. thaliana or in other genomes. In addition, B. depauperata differentially upregulates genes that are involved in the ER stress response. Finally, there are also significant differences in the patterns of differential expression of the photosynthetic genes between the highly thermotolerant B. depauperata and the less tolerant, but better studied A. thaliana.

Materials and Methods

Plant growth and heat stress experiments:

Boechera depauperata and Arabidopsis thaliana (Columbia ecotype) were grown from seed at control conditions and Draftthen subjected to heat shock. Plant growth, heat stress, and RNA isolation methods were as previously described (Gallas and Waters

2015; Halter et al 2017). Over the last 20-30 years, numerous thermotolerance experiments with Arabidopsis have been conducted. Almost all of these studies have examined either Basal Heat stress, Acquired Heat stress, or both (Larkindale et al.

2005; Larkindale and Vierling 2008; Yeh et al 2012). Basal Heat stress (BHS) is defined as a sudden stress from control (approx. 22°C) to heat stress conditions (38°C or above) (Yeh et al. 2012). This type of stress measures the inherent tolerance to stress, i.e., stress tolerance that is not induced but always present. The second major type of stress tolerance is Acquired Heat stress or (AHS). This is examined by providing a moderate heat stress, usually, 38°C for one to two hours, followed by an hour or two of recovery, followed by more extreme stress (above 42°C) for two to four hours (Yeh et al.

2012). AHS experiments examine the impact of the induction of the heat stress

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response genes on the ability of plants to tolerate higher temperatures. In all plants

studied to date, there is a higher tolerance to AHS than to a comparable BHS stress.

That is, a BHS of 43°C might be lethal, but an AHS treatment of 43°C that follows a pre-

treatment of 38°C is not lethal. The conditions chosen for this study were based on

previous studies of Boechera and Arabidopsis basal and acquired thermotolerance

(Gallas and Waters 2015; Halter et al. 2017). The BHS temperature (38°C) was chosen

because this is the most common BHS used and it is known to induce the protective

HSPs in Arabidopsis. The AHS temperature was chosen because it is known to induce

stress in Arabidopsis and in some species of Boechera (Gallas and Waters 2015; Halter

et al. 2017). These temperatures are often used in Arabidopsis studies. One of our

goals is to compare the HS responseDraft of Boechera to Arabidopsis and using

temperatures that are standard for this type of study allows this study to be more easily

compared to those in the thermotolerance literature.

Briefly, two heat shock protocols were applied. 1) basal heat stress (BHS) in

which plants were moved from control temperatures (22°C) and then subjected to 38°C

for two hours. 2) Acquired heat stress (AHS) in which plants were subjected to 38°C for

one hour and then returned to control conditions (22°C) for a two-hour recovery period,

and then finally subjected to heat stress at 43°C for two hours (Halter et al. 2017). Leaf

tissue was harvested and flash-frozen in liquid nitrogen RNA immediately following the

heat stress treatments. Experiments were conducted in triplicate. RNA was isolated as

previously described (Halter et al. 2017). Purified mRNA was then used to construct

RNAseq libraries. These libraries were then sequenced and analyzed as described

below.

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Transcriptomic analysis of Arabidopsis heat stress samples was compared to the known Arabidopsis genome (TAIR 10). All gene and protein names are based on the

Arabidopsis thaliana naming conventions found at The Arabidopsis Information

Resource site (https://www.arabidopsis.org/). However, in order to analyze the RNAseq datasets from Boechera depauperata, a reference transcriptome was generated. The transcriptome assembly RNAseq libraries were prepared from a range of life stages at control conditions, and from both control and heat stressed (basal and acquired) tissue with the Illumina TruSeq protocol and normalized with Illumina Duplex-Specific

Thermostable Nuclease (P/N 15014673 Rev. C). The library was sequenced on an

Illumina Hi-Seq 2000 producing 2 x 94 million100bp paired-end reads. RNA-seq libraries were constructed with IlluminaDraft TruSeq (Illumina, San Diego, CA, USA) libraries.

Barcoded libraries from Arabidopsis thaliana and Boechera depauperata were sequenced in a single lane on separate runs of an Illumina Hi-Seq 2000. Sequencing was performed at The Scripps Research Institute (La Jolla, California, USA). All sequences generated and analyzed here are deposited at the NCBI GEO database under accession number (GSE107820).

In summary, there were two sets of RNAseq sequences analyzed: A) RNAseq libraries from B. depauperata tissue (control, heat stress from both seedling and mature plants) that were used to build a de novo B. depauperata transcriptome, and B) RNAseq libraries from B. depauperata and A. thaliana seedlings for control, after BHS, and after

AHS that were used to examine gene expression induced by heat stress.

Statistical and sequence analysis

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A) Boechera depauperata transcriptome assembly: Raw reads were evaluated with

FastQC (v0.11) trimmed with Skewer (v0.1.120) (Jiang et al. 2014) until a minimum

Phred Q score > 5 was reached. Reads longer than 25bp and with average quality

score Q > 15 were retained. The trimmed paired reads were then assembled de novo

using Trinity (release 2014_07_17) to produce the transcriptome FASTA file. The

completeness of the assembled transcriptome was evaluated with the Core Eukaryotic

Genes Mapping Approach (CEGMA) (Parra et al. 2007). CEGMA identified highly

conserved eukaryotic genes and identified a set of 248 core eukaryotic genes expected

to be expressed in any eukaryotic genome. The CEGMA database was developed from

six model organisms (Homo sapiens, Drosophila melanogaster, Arabidopsis

thaliana, Caenorhabditis elegans, SaccharomycesDraft cerevisiae and Saccharomyces

pombe). The core genes or CEGs were classed into four groups based on the degree of

conservation expected across diverse taxa. The CEGs from group one are expected to

have the lowest level of conservation across taxa and those from group four are

expected to have the highest. A high percentage of coverage of core genes in a de

novo assembly indicates the assembly has complete or near-complete coverage of the

organism’s known biological transcriptional requirements.

Functional annotation of the de novo transcriptome assembly was done using

Blast2GO (version 3.0) (Conesa and Gotz 2008). Blast2GO annotates novel sequences

with gene ontology terms derived from BLAST hits to annotated non-redundant protein

sequences at NCBI. Top 20 hits (by lowest E-value) with a minimum E-Value < 1E-3

were retained. The transcriptome FASTA file and the BLAST query results were

imported to Blast2GO and the transcriptome was annotated with the following

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parameters: E-value hit filter < 1E-3, annotation cut-off (default =55), HSP-hit coverage cutoff (default = 0), and GO weight (default =5). In addition, InterProScan (Jones et al.

2014) was run from within Blast2GO to identify conserved domains in sequences and the retrieved GO terms were merged. ANNEX (Myhre et al. 2006) was run from within

Blast2GO to apply additional GO terms based on known relationships between

Molecular Function annotations and corresponding Biological Process and Cellular

Component annotations. Annotations were validated using the Validate Annotations tool and GO terms were filtered to remove sequences incompatible with land plants (taxa:

3193, Embryophyta). The gene names for Boechera depauperata are based on the de novo transcriptome and have the format “cXXXX-gXX”. Whenever possible we have annotated the Boechera depauperataDraft sequences with the A. thaliana gene number or that of other known genes in NCBI. Genes that have the annotation, “no known homolog” did not have a homolog in the NCBI database. Genes that are

“uncharacterized” have a homolog, but no function can be assigned at this time.

B) Transcriptomics of heat stressed tissue: In order to evaluate differential heat stress gene expression in Boechera and Arabidopsis RNA-Seq reads were mapped using CLC Genomics Workbench. The RNA-seq reads from plants at control, basal heat shock, and acquired heat shock were imported and mapped to the appropriate reference. For Boechera depauperata the Blast2GO annotated FASTA file was imported and reads were mapped to the de novo transcriptome. Arabidopsis thaliana reads were mapped to the TAIR10 reference genome (https://www.arabidopsis.org). For both species, the default read map settings were used: mismatch cost = 2, insertion

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cost = 3, deletion cost = 3, length fraction = 0.8, similarity fraction = 0.8, global

alignment = no, strand-specific = both, maximum number of hits for a read = 10,

expression value = total counts and calculate RPKM for genes without exons. More than

98% of Arabidopsis reads that survived quality control mapped to the reference

Arabidopsis genome, including approximately 15% that mapped to intergenic regions.

More than 96% of Boechera reads that survived quality control mapped to the de novo

transcriptome.

Mapped reads from control, basal HS, and acquired HS were tested for

differential expression using the “Empirical Analysis of DGE” tool with parameters: total

count filter cutoff = 5.0, estimate tagwise dispersions, exact test comparisons against all

pairs, and include FDR corrected P-values.Draft Each test was also performed using edgeR

(v3.16.5) (Nikolayeva and Robinson 2014; Robinson et al. 2010) which uses the same

algorithm as the CLC Workbench and the results were identical. A Benjamini-Hochberg

false discovery rate (Reiner et al. 2003) (FDR) P-value < 0.05 was chosen as the

threshold to identify differential expression.

The software GoSeq (version 1.22.0) (Oshlack et al. 2010) was used to test for

GO term enrichment in differentially expressed genes in each experiment as previously

identified with the EDGE test. For the probability weighting function the length of the

transcript (B. depauperata), or the gene model coding sequence length (A. thaliana)

were used for the probability weight. The Sampling method was used with 1000

iterations. Gene ontology terms with an FDR p-value < 0.05 were accepted as over- or

under-enriched. Because of the extremely sensitive nature of transcriptomics

experiments, a minimum threshold number of reads was imposed. Genes that were

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highly upregulated in terms of fold change but not expressed with the minimum read count were not considered.

Previous studies have shown that Boechera is better able to protect photosynthesis during heat stress than is Arabidopsis. In order to examine differential expression of the photosynthetic pathways during heat stress in these two species, we used the programs Mapman (V. 3.51) and Mercator (Lohse et al. 2014; Bolger et al.

2014). To ensure that we could compare the results across these species we used

Mercator to annotate each Boechera gene by its Arabidopsis homolog. The Boechera transcriptome with Arabidopsis gene ids and the Arabidopsis genome were used as the reference genome data for the pathway analysis within Mapman. Draft Results

Boechera transcriptome assembly:

The Boechera transcriptome was assembled into 82,733 contigs containing

80,628,428 bases. The average contig length was 974 base pairs (bp), the assembly

N50 length was 1,548 bp, and GC content was 43.6% (Table 1). The recovery of core genes in the Trinity assembly was high with 245 of 248 genes at least partially matched

(Table 2). The high rate of recovery of the core genes in the transcriptome indicates that it is a good representation of the genes expressed in Boechera under both control and heat stress conditions. The genes in group one are expected to have the lowest level of conservation, and group four is expected to have the highest. Here (Table 2) we see that all groups have high conservation (above 90%). The two missing groups include inorganic Ion transport and GTPases XPA which interacts with DNA repair protein XPA.

These genes are not known to play a significant role in plant heat stress.

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Differential gene expression during heat stress:

Analysis of RNAseq data indicates similar patterns in the number of genes that

are up and down-regulated during both basal and acquired heat stress in A. thaliana

and B. depauperata (Figure 1). After BHS, A. thaliana upregulated 3,926 genes and

2,012 of these genes were also upregulated after AHS. In A. thaliana 1,906 genes were

only upregulated during BHS. After AHS 2,793 genes were upregulated.

This analysis also revealed that compared to control gene expression patterns 3,832 A.

thaliana genes were down-regulated after BHS and that 2,909 genes were down-

regulated after AHS. Draft

A similar but not identical pattern of gene expression after both heat stress

treatments was seen in B. depauperata. After BHS 2,488 genes were significantly

upregulated in B. depauperata and 3,620 genes were significantly upregulated after

AHS (Figure 1). There were fewer genes significantly down-regulated after BHS

compared to A. thaliana (1,477 genes). However, more genes were significantly down-

regulated in B. depauperata after AHS compared to A. thaliana (2,455 genes). In B.

depauperata only 718 genes were significantly down-regulated after both basal and

acquired HS. This is less than one half the number of genes significantly down-

regulated in A. thaliana after both stresses.

Important differences in the HS responses of A. thaliana and B. depauperata

become apparent when the specific genes that are up and down-regulated are

examined. The analysis of the genes upregulated in response to BHS (Table 3)

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indicates that, as expected, many of the genes induced by BHS in the model plant A. thaliana are the well-studied heat shock genes. For example, of the 50 most upregulated BHS genes (Table 3) 26 are heat shock genes. A distinct and species- specific pattern of heat stress gene expression is found in the highly thermotolerant B. depauperata. Analysis reveals far fewer of the most highly differentially expressed genes induced by BHS in B. depauperata are the heat shock genes (Table 3). Only 11 of the B. depauperata genes are classified as HS genes and most of those are not shared with A. thaliana. A similar pattern can be seen when the genes induced after

AHS are examined (Table 4). From this analysis, it is clear that in A. thaliana19 of the

50 most upregulated AHS genes are heat shock genes (Table 4). Only (6/50) of B. depauperata genes induced after AHSDraft are HSPs (Table 4).

Our data reveals that the vast majority of the genes induced by both basal and acquired heat stress in B. depauperata are not members of the well-studied and characterized Heat Shock Protein families (Tables 3, 4). Most importantly, many of the genes induced by both BHS and AHS in B. depauperata lack homologs in Arabidopsis or other plant species and those that do have homologs have no known function (Table

3 and 4). It is also interesting that one gene that is shared across both HS treatments in both species is galactinol synthase. Galactinol is an osmoprotectant and the gene for galactinol synthase is known to be induced by a variety of stresses including heat

(Sengupta et al. 2015; Jing et al. 2018). Notably, many of the genes that are induced by heat stress in Boechera are involved in the ER-stress response (i.e., e3 ufm1-protein ligase 1; tunicamycin induced protein 1).

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The response of the photosynthetic pathway genes in B. depauperata to both

basal (Figure 2) and acquired HS (Figure 3) is quite distinct from that of A. thaliana

(Figure 2). In Figure 2 it is clear that while BHS results in the downregulation of many A.

thaliana photosynthetic genes. In B. depauperata after BHS most genes either have no

change in expression or exhibit increases in expression (Fig. 2). For example, two

photosystem II genes (homologs of At1g05385 and At1g5140) have higher gene

expression after BHS in B. depauperata. Further, the B. depauperata genes for CPN60

(homologs of At2g28000, At3g13470) also have much higher gene expression after

BHS. A. thaliana does not share this pattern of photosynthetic gene expression after

BHS (Fig. 2). The upregulation of these genes could play a pivotal role in the ability of

B. depauperata to protect photosynthesisDraft during heat stress.

Examination of the changes in gene expression of photosynthetic genes after

AHS shows similar species-specific patterns (Figure 3). AHS results in the

downregulation of a wide array of A. thaliana photosynthetic genes. It is notable that

after AHS in A. thaliana a large number of genes involved in the light reactions exhibit

significantly decreased gene expression (Fig. 3A). However, after AHS, many B.

depauperata genes involved in both the light and dark reactions are significantly

upregulated after AHS (Fig. 3B). The genes for Photosystem BP-2 (homolog of

At2g30790) have increased expression after AHS. Perhaps most notably, the genes

coding for the CPN60Beta proteins (homologs of At3g13470, At1g55490, At3g13470)

exhibit significantly much higher gene expression compared to control (Figure 3B).

Discussion

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This work was undertaken to better understand previous findings that B. depauperata possesses high organismal thermotolerance and can protect photosynthesis at high temperatures (Gallas and Waters 2015; Halter et al. 2017).

Experiments with A. thaliana and B. depauperata were conducted at the same time in the same chambers with identical methods. This was done to allow direct comparisons of B. depauperata with the well-studied A. thaliana. We have found that under these identical heat stress conditions Arabidopsis thaliana and Boechera depauperata, have distinct gene expression patterns. Not unexpectedly, in A. thaliana a wide array of the heat shock genes are expressed under heat stress (Tables 3-4). In particular, a number of the well-studied small heat shock protein genes (Waters 2013) are upregulated during both basal and acquired heatDraft stress in A. thaliana. Notably, the patterns of gene expression in B. depauperata are largely different from that of A. thaliana (Tables 3-4).

Our findings here, that the heat shock genes including the well-characterized small heat shock genes do not play an important role in the heat shock response of B. depauperata, while surprising considering the large body of research conducted in A. thaliana, is consistent with our previous work in Boechera (Gallas and Waters 2015;

Halter et al. 2017). From this, we can conclude that when exposed to identical conditions B. depauperata is not activating the heat shock transcription factors to the same extent as is A. thaliana. This raises the question of how Boechera “feels the heat”.

The cellular signals that transmit the heat stress signal and alter gene expression have been highly studied in Arabidopsis (Mittler et al. 2012) and our findings indicate

Boechera has a distinct and different genomic response to heat stress. If B. depauperata were activating the HSFs we would expect to see a large number of heat

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shock protein genes expressed, i.e. the pattern of gene expression that is demonstrated

here and elsewhere for A. thaliana (Scharf et al. 2012). That the HSPs are not strongly

upregulated suggests that in response to these temperatures B. depauperata might be

activating a distinct set of transcription factors. Future detailed studies of transcriptional

networks during heat stress will be necessary to fully understand the stress-induced

transcriptional networks active in B. depauperata.

Novel genes and genes coding for uncharacterized proteins dominate the heat

shock response of B. depauperata. Many of the genes upregulated in response to both

basal and acquired HS in B. depauperata are either found in other plant genomes, but

are of unknown function; or are novel or orphan genes with no known homologs in other

plant genomes (Tables 3, 4). It is wellDraft established that novel genes can evolve from

non-coding regions, and that these novel or orphan genes play important roles in

lineage-specific adaptations to novel environments (Tautz and Domazet-Loso 2011;

Arendsee et al 2014; Schlötterer 2015). The finding here that the heat shock response

of the highly thermotolerant Boechera depauperata includes numerous novel genes

suggests that these genes are crucial for B. depauperata’s thermotolerance. When the

complete genome of B. depauperata and it’s close relatives are available a more

complete analysis of the origin of these novel genes can take place. Functional analysis

will be needed to determine if the novel genes are responsible for the high

thermotolerance found in B. depauperata (Halter et al. 2017).

Not all of the heat-induced B. depauperata genes are novel or uncharacterized.

Some of the B. depauperata heat-induced genes that have a known function are part of

the unfolded protein and ER- stress response pathways. One of the most highly

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upregulated genes in B. depauperata is a gene for UFM1 ligase (an ortholog of

At3g46220). This is an intriguing finding. UFM1 is a protein with a ubiquitin-like fold.

However, very little is known about the targets or role of this protein in plants (Vierstra

2012). Work done in human cells suggests that UFM1 has a role in apoptosis and in ER stress (Tatsumi et al. 2010; Lemaire et al. 2011). Another gene that is highly heat- induced in B. depauperata codes for Tunicamycin-induced protein (TIN1, an ortholog of

AT5G64510). This gene is known to be induced by ER stress and other abiotic stresses

such as salt (Crosti et al. 2001; Iwata et al. 2010; Iwata and Koizumi 2012; Ozgur et al.

2014). The induction of both UFM1-ligase and TIN1 suggests that in B. depauperata

parts of the ER-stress and Unfolded protein response (UFR) pathways may be

important for the acquisition of thermotoleranceDraft in B. depauperata.

ER stress and the UFR pathways can be induced by a buildup of unfolded

proteins and this can be caused by a variety of stresses including abiotic stresses such

as heat stress (Che et al. 2010; Deng et al. 2013). The UFR is a complex response that

increases both protein degradation and folding capacity in the ER, thus reducing the

number of unfolded proteins in the cell (Vitale and Boston 2008; Howell 2013; Bao and

Howell 2017). ER stress can result in autophagy. During autophagy cell components

including organelles are sent to the lysosome for degradation (Liu and Howell 2016;

Yang et al. 2016). This process recycles damaged cell components and has been

described as a “cell-sparing process” (Howell 2013). Recently a number of publications

have focused on the roles of diverse E3 ligases on the control of chloroplast quality and

autophagy (Woodson et al. 2015; Woodson 2016, 2019; Otegui 2018; Zhang et al.

2018; Peng et al. 2019). Here we have demonstrated that components of both the ER

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stress and UFR pathway are upregulated in B. depauperata during heat stress. While

only parts of these pathways are upregulated, our findings suggest that it is possible

that chloroplast autophagy may play a role in the HS response of B. depauperata. At

this time our data is only suggestive, but it does point a direction towards future studies.

Further experimental studies will need to be conducted to determine the physiological

importance of the transcriptomic data presented here.

The photosynthesis pathways displayed in (Figure 2,3) reveal major differences

between the heat stress response in Arabidopsis and B. depauperata. It has been

previously reported that B. depauperata can maintain high levels of chlorophyll fluorescence during heat stress (GallasDraft and Waters 2015; Halter et al. 2017). It is well established that photosynthesis is highly sensitive to heat stress and that both

Photosystem II and Rubisco Activase are two of the most heat-sensitive components of

the photosynthetic machinery (Salvucci et al. 2001; Sharkey 2005; Zhang and Sharkey

2009; Sharkey and Zhang 2010). When genes associated with the carbon-fixation

reactions were examined it is clear that CPN60BETA2 (Chaperonin 60) is highly

upregulated in Boechera under both 38°C and 43C stress conditions (Fig. 2B, 3B). We

know that Rubisco Activase (RCA) is highly sensitive to heat stress and may be one of

the limiting factors in photosynthetic activity during heat stress (Salvucci et al. 2001).

Further, it is known that CPN60 has been shown to protect RCA during heat stress

(Ciarbelli et al. 2008). The genes for CPN60 were not differentially regulated in

Arabidopsis under either basal or acquired heat stress (Fig. 2A, 3A). This suggests that

Boechera is using CPN60 to differentially protect RCA during heat stress which in turn

may allow photosynthesis to continue during heat stress conditions.

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Boechera depauperata is also differentially regulating genes associated with the

light reactions of photosynthesis during heat stress. Specifically, we have shown that

during both basal and acquired HS B. depauperata upregulates the gene for

Photosystem II subunit P-2 (PSBP-2: At2g30790) (Figure 3). As with CPN60, the genes

for PSBP-2 are not differentially expressed during heat stress in A. thaliana (Fig. 2A,

Fig. 3A). It is well known that PsbP2 is a crucial component of Photosystem II and that it

is required for the proper formation of thylakoid membranes and for photoautotrophy (Yi

et al. 2007; Ido et al. 2009; Roose et al. 2016). PSII is a multi-subunit complex that

initiates electron transport in the chloroplasts and is highly sensitive to damage due to

light (photoinhibition). When this occurs PSII repair requires new components and thus

increased gene expression (NickelsenDraft and Rengstl 2013; Xu et al. 2020). The results

presented here strongly suggest that further detailed studies of the photosynthetic

machinery in Boechera during high-temperature stress should be focused on the process of PSII repair during heat stress.

In conclusion, analysis of RNAseq libraries generated from basal and acquired heat stressed tissue from Arabidopsis thaliana and Boechera depauperata revealed that these two species (both members of the Brassicaceae) have highly distinct and largely non-overlapping heat stress responses. As expected the heat stress response of A. thaliana is largely composed of the heat shock proteins, especially the small heat shock proteins. The heat stress response of B. depauperata is dominated by novel genes,

genes of unknown function, PSBP-2 genes, CPN-60 genes, as well as genes that are

part of the ER and/or unfolded protein responses. Together these findings suggest that

B. depauperata has evolved a unique response to heat stress that combines novel

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genes and parts of existing pathways. This novel combination may be responsible for B.

depauperata’s ability to withstand high-temperatures and protect photosynthesis during

heat stress. Further study of the heat stress response in Boechera depauperata could

open new pathways towards our understanding of how plants can adapt to high

temperature stress and may assist in developing heat-resistant crops.

Acknowledgements

We would like to thank the editors of Botany and two anonymous reviewers who

provided extremely helpful comments and suggestions on previous versions of this

manuscript. This work was supported by a grant from the National Science Foundation

(IOS 0920611) to E.R. Waters. Ian DraftPierce was partially supported by a National

Science Foundation (DUE 0966391) Bioinformatics Statistical Informatics Scholarship.

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Figure 1. Overlap genes that are differentially expressed under basal and acquired heat-stress. Values are shown for A. Arabidopsis thaliana (A.t) and B. Boechera depauperata (B.d). Basal Heat-stress Up-regulated Genes (BHS-U). Basal Heat-stress Down-regulated Genes (BHS-D). Acquired Heat-stress Up-regulated Genes (AHS-U). Acquired Heat-stress Down-regulated Genes (AHS-D).

Figure 2. Impact of basal heat-stress on the expression of genes in photosynthetic Pathways. Each box represents a differentially expressed genes. Boxes shaded blue are upregulated genes, red are downregulated. The relative intensity of the colors indicates the intensity of differential expression compared to control. Plotted values are EDGE test adjusted fold changes, the scale is in the top left corner. A. Arabidopsis thaliana. B. Boechera depauperata.

Figure 3. Impact of acquired heat-stress on the expression of genes in photosynthetic pathways. Each box represents a differentially expressed genes. Boxes shaded blue are upregulated genes, red are downregulated. The relative intensity of the colors indicates the intensity of differential expression compared to control. Plotted values are EDGE test adjusted fold changes, the scale is in the top left corner. A. Arabidopsis thaliana. B. Boechera depauperata. Draft

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Table 1. Summary statistics of B. depauperata transcriptome de novo assembly

Parameter Trinity assembly

Input reads 94,662,505

Mapped reads 73,734,639 Reads not mapped 20,929,437Draft Number of contigs 82,733

Contig N50 (base pairs) 1,548

Contig average length 974 (base pairs)

Total assembled bases 80,628,428

Percent GC 43.6

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Table 2. Core Eukaryotic Genes in B. depauperata transcriptome. CEG Groups Genes recovered Percent recovered

Group 1 (n=661) 65 97%

Group 2 (n=56*) 52 98%

Group 3 (n=61*) 60 93%

Group 4 (n=65*) 64 98%

Total (n=2482) 241 97%

1The total number of Core Eukaryotic Genes (CEGs)Draft in each group. 2All 248 CEGs are characterized as house-keeping gene families and are expected to be present in all eukaryotic genomes.

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Table 3. Genes with highest change in expression after a Basal Heat Stress (BHS).

Arabidopsis thaliana Boechera depauperata Gene Annotation TAIR ID Fold Change Gene ID Fold Change Gene Annotation AT3G46230 16.23 heat shock protein 17.4 c33426_g1_i5 53.11 galactinol synthase 1 AT4G10250 16.05 HSP20-like chaperones superfamily protein c35686_g1_i8 47.24 e3 ufm1-protein ligase 1 homolog AT1G52560 15.62 HSP20-like chaperones superfamily protein c30323_g1_i3 35.82 no known homolog AT4G27670 15.59 heat shock protein 21 c26432_g1_i8 34.95 no known homolog AT5G12030 15.38 heat shock protein 17.6A c35686_g1_i7 34.25 e3 ufm1-protein ligase 1 homolog AT1G59860 15.38 HSP20-like chaperones superfamily protein c34550_g3_i1 31.91 developmentally-regulated g-protein 2 AT5G59720 15.32 heat shock protein 18.2 c32523_g1_i1 29.13 hsp70-binding protein 1-like AT2G29500 15.12 HSP20-like chaperones superfamily protein c26432_g1_i9 27.56 no known homolog AT5G48570 14.82 FKBP-type peptidyl-prolyl cis-trans isomerase family protein c33010_g1_i1 27.42 no known homolog AT5G25450 14.78 Cytochrome ubiquinol oxidase c29978_g1_i2 27.00 l-ascorbate peroxidase cytosolic-like AT1G07400 14.78 HSP20-like chaperones superfamily proteinDraftc29978_g1_i11 25.44 l-ascorbate peroxidase cytosolic-like AT1G71000 14.76 Chaperone DnaJ-domain superfamily protein c31317_g1_i7 24.88 hsp70-hsp90 organizing protein 3-like AT1G53540 14.75 HSP20-like chaperones superfamily protein c22536_g1_i2 24.49 uncharacterized protein LOC104722666 AT1G17870 14.69 ethylene-dependent gravitropism-deficient yellow-green-like 3 c34173_g4_i2 24.49 heat shock protein 83-like AT1G16030 14.65 heat shock protein 70B c32384_g1_i13 24.20 pentatricopeptide repeat-containing protein AT5G54165 14.60 no known homolog c26432_g1_i6 22.90 no known homolog AT3G24500 14.56 multiprotein bridging factor 1C c34777_g1_i4 22.38 zinc finger protein zpr1-like AT1G72660 14.52 P-loop containing nucleoside triphosphate hydrolases c30928_g1_i4 22.33 no known homolog AT3G12580 14.52 heat shock protein 70 c32515_g1_i10 22.30 ultraviolet-b receptor uvr8-like AT2G20560 14.52 DNAJ heat shock family protein c32962_g1_i5 21.95 atp-dependent zinc metalloprotease ftsh chloroplastic AT5G37670 14.52 HSP20-like chaperones superfamily protein c31317_g1_i1 20.59 hsp70-hsp90 organizing protein 3 AT1G54050 14.49 HSP20-like chaperones superfamily protein c26737_g1_i7 20.25 synaptotagmin-4-like isoform x1 AT5G51440 14.47 HSP20-like chaperones superfamily protein c26050_g1_i2 20.07 inactive rhomboid protein 1-like AT1G74310 14.46 heat shock protein 101 c30491_g1_i2 19.81 no known homolog AT4G25200 14.46 mitochondrion-localized small heat shock protein 23.6 c29245_g1_i2 19.59 protein tunicamycin induced 1 AT2G32120 14.45 heat-shock protein 70T-2 c30904_g5_i3 19.14 sHSP class I heat shock AT5G12020 14.37 17.6 kDa class II heat shock protein c24839_g1_i1 19.12 sHSP heat shock protein AT2G47180 13.92 galactinol synthase 1 c34777_g1_i3 18.99 zinc finger protein zpr1-like

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AT4G12400 13.89 stress-inducible protein c34655_g1_i1 18.83 peptidyl-prolyl cis-trans isomerase fkbp65 AT2G46240 13.87 BCL-2-associated-6 c34843_g1_i3 18.75 chaperone protein 1 AT5G07330 13.82 no known homolog c32962_g1_i8 18.48 atp-dependent zinc metalloprotease ftsh chloroplastic-like AT2G26150 13.75 heat shock transcription factor A2 c26050_g1_i3 18.45 inactive rhomboid protein 1-like AT5G52640 13.50 heat shock protein 90.1 c26572_g1_i2 18.18 cytochrome b-c1 complex subunit 7-2 AT5G12110 13.48 Glutathione S-transferase c32505_g1_i9 18.17 calcyclin-binding AT1G21550 13.40 Calcium-binding EF-hand family protein c34173_g4_i1 18.12 heat shock protein 83-like AT1G03070 13.01 Bax inhibitor-1 family protein c32505_g1_i2 17.86 calcyclin-binding AT3G10020 12.58 no known homolog c28228_g1_i1 17.80 small heat shock chloroplastic-like AT3G09350 12.56 Fes1A c22147_g1_i1 17.42 sHSP kda heat shock peroxisomal AT1G30070 12.50 SGS domain-containing protein c27663_g1_i1 17.41 heat stress transcription factor a-7a-like AT5G01180 12.38 peptide transporter -5 c35686_g1_i4 17.26 e3 ufm1-protein ligase 1 homolog AT5G47830 12.34 no known homolog c22392_g1_i2 17.10 no known homolog AT5G09590 12.26 mitochondrial HSP70-2 c30904_g5_i2 17.09 sHSP class I heat shock AT4G23493 12.21 no known homolog Draftc9792_g1_i1 17.08 sHSP class I heat shock protein AT5G10695 12.15 no known homolog c35686_g1_i3 17.03 e3 ufm1-protein ligase 1 homolog AT3G08970 11.96 DNAJ heat shock N-terminal domain-containing protein c33819_g1_i3 16.99 ubiquitin-associated translation elongation factor ef1b protein AT2G19310 11.93 HSP20-like chaperones superfamily protein c29978_g1_i6 16.89 l-ascorbate peroxidase cytosolic AT2G37180 11.78 Aquaporin-like superfamily protein c30904_g5_i1 16.87 class I heat shock AT3G53230 11.67 ATPase c21290_g1_i1 16.77 pentatricopeptide repeat-containing protein AT1G67360 11.60 Rubber elongation factor protein (REF) c30904_g5_i4 16.65 sHSP class I heat shock AT3G28210 11.43 zinc finger (AN1-like) family protein c25637_g1_i1 16.63 sHSP heat shock mitochondrial

A Benjamini-Hochberg false discovery rate (Reiner, Yekutieli et al. 2003) (FDR) p-value < 0.05 was chosen as the threshold to identify differential expression. Genes in bold are members of the well-studied heat shock protein families.

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Table 4. Genes with highest change in expression after an Acquired Heat Stress (AHS).

Arabidopsis thaliana Boechera depauperata Fold Gene Annotation Fold TAIR ID Gene ID Gene Annotation Change Change AT1G56600 23.00 galactinol synthase 2 c35942_g1_i4 200.42 retrovirus-related pol polyprotein AT5G07330 22.22 no known homolog c19645_g1_i2 164.85 cation h(+) antiporter 18-like AT5G52630 18.56 mitochondrial RNAediting factor 1 c33426_g1_i7 146.75 galactinol synthase 1 AT1G71000 18.04 Chaperone DnaJ-domain superfamily protein c35942_g1_i2 130.87 no known homolog AT5G05410 17.66 DRE-binding protein 2A c29988_g4_i1 119.85 uncharacterized protein AT4G37990 17.42 elicitor-activated gene 3-2 c31317_g1_i7 119.64 hsp70-hsp90 organizing protein 3-like AT5G37670 17.02 HSP20-like chaperones superfamily protein c30471_g1_i4 119.14 probable glucose-1-phosphate AT1G52560 16.22 HSP20-like chaperones superfamily protein c29455_g1_i1 111.98 heat shock 70 kda protein 8 AT4G27657 16.02 no known homolog c34655_g1_i4 99.92 peptidyl-prolyl cis-trans isomerase fkbp62-like AT5G15960 15.32 stress-responsive protein (KIN1) / stress-induced protein (KIN1) c19645_g1_i1 94.80 cation h(+) antiporter 18-like AT5G59720 14.05 heat shock protein 18.2 Draftc33410_g1_i2 94.37 uncharacterized vacuolar membrane protein yml018c AT5G12030 13.87 heat shock protein 17.6A c33426_g1_i1 94.32 galactinol synthase 1-like AT2G29500 13.75 HSP20-like chaperones superfamily protein c31166_g1_i4 89.05 no known homolog AT2G22240 13.46 myo-inositol-1-phosphate synthase 2 c27782_g2_i1 86.42 gdsl esterase lipase 3-like isoform x2 AT5G47610 13.03 RING/U-box superfamily protein c34655_g1_i9 85.45 peptidyl-prolyl cis-trans isomerase fkbp62-like AT3G28550 12.63 Proline-rich extensin-like family protein c34173_g4_i2 79.98 heat shock protein 83-like AT1G33350 12.59 Pentatricopeptide repeat (PPR) superfamily protein c24401_g1_i1 77.69 no known homolog AT2G20560 12.38 DNAJ heat shock family protein c33496_g1_i6 74.28 probable zinc metallopeptidase chloroplastic AT5G03210 12.06 no known homolog c28380_g1_i1 71.35 no known homolog AT5G62020 11.86 heat shock transcription factor B2A c32523_g1_i1 70.90 hsp70-binding protein 1-like AT2G46240 11.54 BCL-2-associated-6 c34655_g1_i1 66.95 peptidyl-prolyl cis-trans isomerase fkbp65 AT5G43170 11.41 zinc-finger protein 3 c26211_g1_i1 66.79 PREDICTED: uncharacterized protein LOC104768939 AT3G54580 11.29 Proline-rich extensin-like family protein c28278_g1_i1 62.99 PREDICTED: uncharacterized protein LOC104728675 AT5G59820 11.24 C2H2-type zinc finger family protein c33426_g1_i5 62.87 galactinol synthase 1 AT5G15250 11.12 FTSH protease 6 c34274_g2_i5 62.01 atp-dependent 6-phosphofructokinase 7-like AT3G03270 11.11 Adenine nucleotide alpha hydrolases-like superfamily protein c40391_g1_i1 61.76 no known homolog AT4G36990 10.97 heat shock factor 4 c35686_g1_i7 58.95 e3 ufm1-protein ligase 1 homolog AT1G53540 10.94 HSP20-like chaperones superfamily protein c34655_g1_i11 58.76 peptidyl-prolyl cis-trans isomerase fkbp65

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AT5G35320 10.82 no known homolog c22000_g1_i1 56.55 no known homolog AT5G54165 10.78 no known homolog c22682_g2_i2 55.38 no known homolog AT3G50970 10.66 dehydrin family protein c13684_g1_i1 55.12 transposon tf2-1 polyprotein isoform x1 AT4G27670 10.64 heat shock protein 21 c34334_g1_i5 53.40 heat shock cognate protein 80-like AT4G36988 10.26 conserved peptide upstream open reading frame 49 c29978_g1_i2 53.33 l-ascorbate peroxidase cytosolic-like AT4G10250 10.17 HSP20-like chaperones superfamily protein c31317_g1_i1 50.88 hsp70-hsp90 organizing protein 3 AT3G12580 9.93 heat shock protein 70 c32384_g1_i13 50.77 pentatricopeptide repeat-containing protein mitochondrial AT3G46230 9.92 heat shock protein 17.4 c35340_g2_i1 50.10 protein sgt1 homolog a-like AT3G15340 9.76 proton pump interactor 2 c34655_g1_i7 48.14 70 kda peptidyl-prolyl isomerase-like AT3G24500 9.62 multiprotein bridging factor 1C c34655_g1_i2 47.51 peptidyl-prolyl cis-trans isomerase fkbp65 AT1G54050 9.56 HSP20-like chaperones superfamily protein c19310_g1_i1 47.04 no known homolog AT1G59860 9.54 HSP20-like chaperones superfamily protein c32962_g1_i6 46.16 vacuolar amino acid transporter 1-like AT1G16030 9.53 heat shock protein 70B c32505_g1_i2 45.69 calcyclin-binding AT5G10695 9.45 no known homolog c32670_g1_i1 45.31 dehydration-responsive element-binding protein 2a-like AT1G07400 9.43 HSP20-like chaperones superfamily proteinDraftc26327_g1_i2 45.27 f1f0-atpase inhibitor protein AT4G25200 9.38 mitochondrion-localized small heat shock protein 23.6 c31166_g1_i3 45.17 no known homolog AT1G74310 9.28 heat shock protein 101 c18072_g1_i1 44.82 mediator of rna polymerase ii transcription subunit 37c AT3G41762 9.26 no known homolog c30973_g2_i1 44.79 protein polar localization during asymmetric division AT4G12400 9.13 stress-inducible protein c35956_g1_i3 44.64 no known homolog AT1G61340 9.13 F-box family protein c23392_g1_i2 44.63 phd finger-like domain-containing protein 5b AT3G10020 9.11 no known homolog c30578_g1_i1 44.11 no known homolog AT1G20440 9.08 cold-regulated 47 c24026_g1_i2 44.10 no known homolog

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Draft

215x279mm (220 x 220 DPI)

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272x99mm (600 x 600 DPI) Draft

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246x88mm (600 x 600 DPI) Draft

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