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Single-base resolution methylome analysis shows epigenetic changes in Arabidopsis seedlings exposed to microgravity spaceflight conditions on board the SJ-10 recoverable satellite

Single-base resolution methylome analysis shows epigenetic changes in Arabidopsis seedlings... www.nature.com/npjmgrav ARTICLE OPEN Single-base resolution methylome analysis shows epigenetic changes in Arabidopsis seedlings exposed to microgravity spaceflight conditions on board the SJ-10 recoverable satellite 1 1 1 1 Peipei Xu , Haiying Chen , Jing Jin and Weiming Cai DNA methylation is a very important epigenetic modification that participates in many biological functions. Although many studies of DNA methylation have been reported in various plant species, few studies have assessed the global DNA methylation pattern in plants challenged by exposure to microgravity conditions. In this report, we mapped the Arabidopsis genome methylation pattern changes associated with microgravity conditions on board the Chinese recoverable scientific satellite SJ-10 at single-base resolution. Interestingly, we found epigenetic differences in Arabidopsis seedlings exposed to microgravity in that the Arabidopsis genome exhibits lower methylation levels in the CHG, CHH, and CpG contexts under microgravity conditions. Microgravity stimulation was related to altered methylation of a number of genes, including DNA methylation-associated genes, hormone signaling related genes, cell-wall modification genes and transposable elements (TEs). Relatively unstable DNA methylation of TEs was responsible for the induction of active transposons. These observations suggest that DNA demethylation within TEs may affect the transcription of transposons in response to microgravity conditions. In summary, the results of this investigation are beneficial for understanding the mechanism of plant adaptation to microgravity and improve strategies to allow plants to adapt to space. npj Microgravity (2018) 4:12 ; doi:10.1038/s41526-018-0046-z INTRODUCTION indicate an association between environmental stresses and DNA methylation alterations. On Earth, life is adapted to a constant gravitational force, and DNA cytosine methylation functions as an important controller biological processes in organisms have evolved under this natural of gene mRNA levels. In the Arabidopsis genome, DNA cytosine constant. Many limiting factors for plant growth under micro- methylation is mostly detected at CpG sites and can also occur at gravity conditions have been identified. Therefore, it is expected CHG and CHH residues (H indicates A, T, or C). In the Arabidopsis that plants have sufficient and sustainable mechanisms that allow genome, most methylation in the gene body is detected at CpG for them to survive under microgravity conditions. To identify sites, while CpG, CHH, and CHG site methylation occurs elsewhere plant responses to the microgravity stimulus, many groups have 12 and in repetitive regions. CG methylation is enriched in TEs and been using various approaches to identify genes with altered 13 gene bodies, but non-CG methylation is mostly present in TEs. 1–5 expression levels under microgravity conditions. The results Often, methylation of gene promoter regions is believed to inhibit have shown that microgravity signals can be transduced into gene expression, while methylation within introns and exons can molecular signaling cascades, which lead to plant adaptation. promote gene transcription. MET1 is the main CG methyltrans- Currently, it is obvious that epigenetic alterations are involved ferase, CMT3 is the main CHG methyltransferase, and DRM2 is the in plant adaptation to environmental stress. A series of current main CHH methyltransferase and is guided by small RNAs. reports have shown that DNA methylation leads to control of gene Epigenetic features play a critical role in controlling gene mRNA levels and plays important roles in abiotic and biotic expression and the subsequent response of an organism to its stresses. For example, drought stress results in a global DNA environment. As a major epigenetic modification, DNA methylation is not directly encoded in the genome sequence, and yet can methylation alteration in rice. Salt stress induces OsMYB91 gene modify expression and may be inherited for at least one expression due to the reduced cytosine methylation level in its generation. Several studies have shown that large numbers of promoter region. In addition, widespread alterations in DNA plant genes are differentially expressed in response to spaceflight. methylation of the poplar genome in response to drought 9 Learning more about the spaceflight methylome of plants will treatment indicate adaption to the local environment. Moreover, contribute to the foundational understanding of how plants adapt epigenetic variations in transposable element (TE) regions can be 10 to spaceflight. In this research, we showed the results of an SJ-10 affected by environmental stresses. Silencing of the ZmMET1 spaceflight experiment. The experiment was a part of the SJ-10 gene after low-temperature treatment leads to demethylation of mission, an experimental project of Chinese Academy of Sciences the Ac/Ds transposon region in maize roots. In sum, these results in April 2016. Arabidopsis seedlings were exposed to spaceflight on Laboratory of Photosynthesis and Environment, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, No. 300 Fenglin Road, Shanghai 200032, China Correspondence: Weiming Cai (wmcai@sibs.ac.cn) These authors contributed equally: Peipei Xu, Haiying Chen. Received: 14 December 2017 Revised: 28 May 2018 Accepted: 1 June 2018 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. board of SJ-10. To evaluate DNA methylome during spaceflight, we other histogram tracks represent differentially methylated regions profiled DNA methylation genome wide in Arabidopsis seedlings. (DMRs) in the CHG (d), CHH (e), and CpG (f) contexts. Whole-genome bisulfite sequencing (BS-Seq) allows DNA methyla- Previous research showing that cytosine methyltransferases and tion to be measured at the whole-genome level with single demethylases play important roles in the maintenance of genomic nucleotide resolution. These results will be very useful for our methylation indicates that these enzymes are involved in a understanding of the potential role of cytosine DNA methylation in number of biological functions. To investigate possible altered plants adapting to the outer space environment. methylation patterns in methyltransferase- and demethylase- related genes in response to microgravity conditions, we examined all of these genes to determine whether there were RESULTS any changes in their DNA methylation levels under microgravity. Experimental design and sample fixation on the recoverable The details of these results for DMT1, which encodes the cytosine scientific satellite SJ-10 methyltransferase MET1, DME, which encodes the DNA glycosy- lase DEMETER, and TET14, a tetraspanin family gene, are shown in The specific equipment used in the space experiment was Table S1. The results indicate that exposure of the plants to designed and constructed by Shanghai Institution of Technical microgravity was accompanied by alterations in the DNA Physics (Fig. 1a, b). The equipment included two cultivation units, methylation patterns of specific genes. a canal system and a pump support system. The polysulfone chambers in the cultivation units had windows that were covered by a gas permeable membrane. The chambers were connected to Altered methylation profiles of functional genes in response to the fixative unit by tube connectors. The Chinese recoverable microgravity conditions scientific satellite SJ-10 was launched at 01:38 on April 6, 2016 and To perform an in-depth analysis of the functional categories of landed at 16:30 on April 18, 2016. The air temperature near the genes that showed alterations in their methylation patterns under incubator averaged approximately 23 °C. The average relative microgravity conditions, we used gene ontology (GO) enrichment humidity near the incubator was approximately 25%, and the to categorize these genes. In the CHG context in the category of −3 microgravity level was estimated to be approximately 10 to biological processes, altered methylation-related (AMR) genes −4 15,16 10 g during the unified flight phase. Seedlings were fixed were enriched for cell wall-related processes, carbohydrate with RNAlater® after growth for 60 h under microgravity condi- metabolic processes, defense response, and nitrogen compound tions. They were harvested approximately 4 h after landing, stored transport. Changes in these biological processes were also shown at 4 °C, and transported to our laboratory for further assays. in transcriptomic studies in several previous plant spaceflight Seedling DNA was extracted and used for methylome analysis. To 3,4 experiments. Although the results of various spaceflight halt cellular activities in space and preserve the DNA methylome experiments differ for a variety of reasons, changes in cell wall- profile, RNAlater® was added to the culture chambers on board SJ- related processes and defense response were consistently 10 and on the ground. RNAlater® has been experimentally verified 4 detected. This suggests that the weakening of the plant cell wall to effectively preserve DNA and to yield high-quality RNA and has that occurs under microgravity may be regulated at the level of been used successfully in spaceflight applications. Samples of DNA methylation. Changes in metabolism-associated AMR genes, the 1 g ground controls in cultivation unit 1 were also fixed in including carbohydrate metabolic processes and nitrogen com- RNAlater® at the same time as the in-space samples (Fig. 1d). pound transport, suggested that energy metabolism might be regulated in response to the microgravity environment. The Global DNA methylation patterns of Arabidopsis seedlings under defense response that occurs in plants during spaceflight might microgravity conditions be due to the changes in forces on the cell wall under microgravity. The discovery that defense response AMR genes To identify the DNA methylation pattern that was present in the are expressed under microgravity may validate this assumption seedlings during spaceflight, single-base resolution methylome (Fig. 3a). In the CHH context in the category of biological analysis was conducted using Arabidopsis (Col-0) seedlings that processes, AMR genes for metabolic processes, cellular metabolic had been grown for 60 h under microgravity conditions. The DNA processes, biosynthetic processes, signaling transduction, methylation profiles at the whole-genome level were determined; oxidation–reduction processes, and lipid biosynthesis were analysis of the read intensity around the annotated genes enriched. Lipid biosynthesis has been implicated in gravity revealed lower DNA methylation levels under microgravity conditions (Fig. 2). Because euchromatin and heterochromatic responses in Arabidopsis, and is likely involved in adjusting the genomic regions such as coding sequences and repetitive polarity of cells. Here, we also found the lipid biosynthesis AMR sequence regions display different DNA methylation patterns, genes. Gravity sensing in plant cells involves signals related to we further evaluated the detailed methylation patterns within oxidative stress. In fact, oxidation–reduction process AMR genes genes, including coding sequences (CDS) and noncoding areas. were also identified. Regarding cellular components such as cell, The results showed that CpG sites had the highest methylation intracellular, organelle, plasma membrane, and cell periphery, levels and that CHH sites had the lowest methylation levels in AMR genes were enriched (Fig. 3b). In the CpG context in the each gene region (Fig. 2a–c). In the CpG context, CpG methylation category of biological processes, AMR genes for mRNA transport, in the 5’UTR and 3’UTR regions was lower than that in coding mRNA export from the nucleus, microtubule-based movement, regions, and the 5’UTR methylation level was much lower than telomere maintenance in response to DNA damage, regulation of that in the 1500-bp upstream region and in the 3’UTR (Fig. 2c). The telomere maintenance and inositol phosphate phosphorylation CHG context appeared to be quite different from the CpG context, were enriched. Because space radiation can cause damage to DNA with much higher methylation levels in 1500-bp upstream regions directly, the radio-adaptive response (RAR) of Arabidopsis root and 5’UTR regions, and CDS displayed the lowest methylation growth is modulated under microgravity conditions, and DNA level (Fig. 2a). However, the CHH context showed higher damage repair in RAR is regulated by microgravity. Here, we methylation levels in the 5’UTR and 3’UTR regions and lower found DNA damage and regulation of telomere maintenance AMR methylation levels in the 1500-bp upstream region and CDS sites genes, supporting this assumption. Inositol phosphate signaling (Fig. 2b). Circos plots show the differences in methylation changes has also been shown to occur in response to microgravity between ground controls (G1–G3) and plants exposed to conditions. For cellular components, AMR genes for the microgravity conditions (S1–S3) (Fig. 2d–f). The outer track functional categories of Cul4-RING ubiquitin ligase complex, indicates the five chromosomes of the Arabidopsis genome. The nuclear envelope, and nuclear pore were enriched (Fig. 3c). npj Microgravity (2018) 12 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. Fig. 1 Hardware and experimental procedures used in the experiment on board the SJ-10 recoverable satellite. a Appearance of the hardware used in the spaceflight experiment. The equipment functioned as a housing for the culture chambers. b Distribution of internal components of the hardware. The hardware includes culture chambers, a pump support system, a canal system and a fixative unit connected to the culture chambers. c Arabidopsis seedlings were grown in culture chambers during the space experiment. d Schematic view of the Arabidopsis seedlings on board the SJ-10 satellite during spaceflight and on the ground. Arabidopsis seedlings in culture chambers were transferred from Petri dishes 3 days before launching. Seedlings in culture chambers installed in cultivation unit 1 were grown for 60 h under microgravity conditions and fixed in space using RNAlater®. The seedlings in the culture chamber installed in cultivation unit two grew for 11 days under microgravity; after this time, they were still alive and returned with the satellite. The g-profile during SJ-10 satellite in orbit is described in the 15,16 references. The g-profile during launch was as follows: the first-level maximum static overload was 4.8 g in flight for 150 s; the second-level maximum static overload was 6.0 g in flight for 180 s Numerous TE genes are hypomethylated under microgravity CpG contexts in the TE regions and methylation levels in the 1-kb conditions upstream and downstream regions were demonstrated (Fig. 4a). TEs presented lower methylation levels in the CHG, CHH, and CpG Previous research has shown that mobilization and silencing of contexts in spaceflight samples. The results showed that a large TEs are often accompanied by disruption of DNA methylation. TEs can affect the size of the genome, create insertions and other proportion of the TEs in these contexts are differentially mutations, and influence gene expression patterns. Under methylated and that hypomethylation of TE sites was likely the microgravity conditions, many differentially methylated TEs were result of the induction of active TEs. demethylated (Fig. 4a). The methylation patterns of the CHG, CHH, Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 12 DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. Fig. 2 DNA methylation patterns in various genomic regions. a–c Distribution of the DNA methylation levels in the CHG a, CHH b, and CpG c contexts among various gene regions, including the promoter, 5’UTR, gene body and 3’UTR in ground controls (G1–G3), and in plants maintained under microgravity conditions (S1–S3). d–f Circos plots showing Arabidopsis genomic methylation in ground controls (G1–G3) and in plants maintained under microgravity conditions (S1–S3). The outer track represents five chromosomes of the Arabidopsis genome. The other histogram tracks represent differentially methylated regions in the CHG d, CHH e, and CpG f contexts Altered methylation profiles of TEs of different lengths in response whereas in the CHH context they were assigned to pathways to microgravity conditions related to metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and ribosomes (Fig. Previous research has shown that the length of TEs can affect their 5b). In the CpG context, they were mainly assigned to pathways methylation levels. Moreover, TE methylation levels can associated with metabolism-related processes (such as sulfur influence transposon expression in response to environmental metabolism, terpenoid backbone metabolism, N-glycan biosynth- stress. To further identify the association between methylation esis, butanoate metabolism, and beta-alanine metabolism), ABC status and TE length in Arabidopsis, TEs were divided into three transporters, nucleotide excision repair, protein procession in the groups: the first group with lengths shorter than 500 bp, second ER, and endocytosis (Fig. 5c). Many of the pathways identified here group with lengths ranging from 500 to 2000 bp; and third group in the studies of transcriptomic analysis under microgravity have with lengths of more than 2000 bp. Compared with the ground 3,4 also been found in other spaceflight experiments. Some of controls (G1–G3), the first group of TEs showed a striking decrease these pathways play important roles in plant gravity perception in CHG and CHH methylation levels under microgravity conditions and in the responses of plants to microgravity. For instance, ABC (S1–S3) (Fig. 4b). The second group showed a marginal decrease transporters have been shown to play a role in the transport of the in the CHG and CHH methylation contexts (Fig. 4c). The third phytohormone auxin. The enrichment of AMR genes encoding group showed a decrease in the CHG context but no decrease in ABC transporters implies that auxin transport can be modulated the CHH or CpG methylation contexts under microgravity by microgravity conditions at the level of DNA methylation. conditions (Fig. 4d). We propose that demethylation of TEs plays We also further characterized methylation alterations in genes an important role under microgravity conditions. encoding transcription factors (TFs). The PlantTFDB predicts that the Arabidopsis genome contains more than 2200 TFs. Approxi- Widespread DNA methylation in response to microgravity mately, 36 TF genes showed altered methylation levels during spaceflight microgravity spaceflight. These included bHLH, bZIP, GATA, WRKY, To identify the potential influence of microgravity spaceflight on and NAC members (Table S3). Previous spaceflight experiments the methylation level of the whole genome, we analyzed the demonstrated that the expression of many TF genes is altered 2,3 DMRs in flight seedlings and ground controls. The distribution and under microgravity. For example, in the TROPI-2 project, an NAC methylation levels of the DMRs of five Arabidopsis chromosomes TF gene, ANAC104/XND1 (At5g64530), was up regulated under are shown in Figs. S3–S6. To determine the molecular functions of microgravity conditions. ANAC104/XND1 regulates the synthesis these DMR-related genes, we conducted KEGG pathway enrich- of tracheal element secondary walls and can affect plant height ment analysis. In the CHG context, these genes were mainly and the length of tracheal elements in stems and hypocotyls. assigned to pathways associated with metabolism (starch and Another NAC TF gene, NAC1 (NAC domain-containing protein 21/ sucrose metabolism, phenylalanine biosynthesis, purine metabo- 22, At1g56010), was down regulated under microgravity. NAC1 lism, glutathione metabolism, amino sugar, and nucleotide sugar encodes a transcription factor that is induced by auxin and metabolism), proteasomes, ribosomes, and phagosomes (Fig. 5a), mediates auxin signaling to promote lateral root development. npj Microgravity (2018) 12 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. Fig. 3 GO-term enrichment network of genes with altered methylation levels in the Arabidopsis genome. GO-term enrichment analysis of altered gene methylation patterns in the CHG a, CHH b and CpG c contexts under microgravity conditions was conducted. The bar represents the number of genes in the test set belonging to each GO category. Only the top 10 GO terms are listed (p < 0.05) Alteration of the expression of auxin-related and cell wall-related under microgravity have been identified, including the genes genes under microgravity is closely related to plant adaptation to encoding auxin responsive factors (ARF1-6, ARF8, ARF19, ARF21, microgravity. Therefore, it is proposed that the possible differential and ARF22), auxin signaling F-box proteins (TIR1, AFB2, and AFB5), methylation of genes encoding TFs may also contribute to the auxin responsive proteins (IAA9 and IAA33), transport protein (BIG) adaptation of plants to microgravity. and indole-3-acetaldehyde oxidases (AAO1 and AAO2). TIR1 Abundant DMRs were found to be involved in the “Signaling” encodes an auxin receptor that mediates auxin-regulated pathway. The observed alterations in the methylation of genes transcription. The observation of altered TIR1 methylation levels involved in hormone signaling pathways were functionally implies that microgravity affects auxin signaling in plants. Aux/ characterized. Genes involved in hormone signaling pathways, IAA proteins are short-lived TFs that function as repressors of early including auxin, ABA (abscisic acid), ethylene, and BRs (brassinos- auxin response genes. Repression is thought to result from the teroids), were also differentially methylated (Table S4). Notably, interaction of the repressors with auxin response factors (ARFs). several auxin-related genes whose DNA methylation was changed Formation of heterodimers with ARF proteins may alter the ability Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 12 DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. Fig. 4 Methylation patterns of TEs and different types of TEs with different lengths in the Arabidopsis genome. a Methylation patterns of TEs. The distribution of the methylation read density for all TEs in the Arabidopsis genome between ground controls (G1–G3), and under microgravity conditions (S1–S3) is shown. The x-axis represents the TE body and its 1-kb upstream and downstream regions. The y-axis indicates the average methylation level. Three types of methylation patterns, CHG, CHH, and CpG, are shown in the figure. b–d Methylation patterns of TEs with different lengths. Methylation patterns of TEs with different lengths in ground controls (G1–G3) and in plants grown under microgravity conditions (S1–S3). TEs were divided into quintiles based on their lengths: the first quintile is the shortest, and the third quintile represents TEs of the longest length. The read density distribution for TEs with lengths of (b) less than 500 bp, (c) 500–2000 bp, and (d) more than 2000 bp within the Arabidopsis genome is shown. Three types of methylation patterns, CHG, CHH, and CpG, are shown in each figure npj Microgravity (2018) 12 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. Fig. 5 Pathway analysis of genes that showed altered methylation. KEGG pathway enrichment analysis of CHG a, CHH b, and CpG c differentially methylated genes in the Arabidopsis genome in plants grown under microgravity conditions and ground controls was performed. The size of the circle represents the gene numbers, and the color represents the q value Fig. 6 Comparison of the phenotypes of Arabidopsis seedlings grown under microgravity and on the ground. a Seedlings that served as ground control samples. b Seedlings that returned to earth with the satellite after growth under microgravity for 11 days. The experimental procedure was the same as described in Fig. 1d. The leaf area was measured using ImageJ. Bar = 1 cm (in a, b). The data shown are the mean values obtained for eight leaves (n = 8); the vertical bars represent the standard deviation. Asterisks indicate a statistically significant difference (p < 0.05) between spaceflight and control samples using Student’s two-tailed t test of these proteins to modulate the expression of genes involved in involved in ABA biosynthesis (ABA3) and brassinosteroid insensi- the early auxin response. The decreased methylation level of ARF tive1 associated receptor kinase (BAK1) were also decreased. genes and the increased methylation level of the IAA9 and IAA33 Alterations in the methylation of genes related to cell wall were genes may contribute to an increased auxin response. The also identified. Hypergravity has been shown to upregulate cell gravimorphogenesis of plant organs was changed due to the wall rigidity in stems and roots. However, plant cell wall rigidity effects of microgravity on the polar transport of auxin. BIG is lower under microgravity conditions than under ground encodes a membrane-associated protein and is required for auxin conditions. The thickness of cell walls decreased in response transport. We found that BIG was hypermethylated in space; thus, to the microgravity stimulus. The space-grown plants also microgravity might inhibit auxin transport by increasing BIG contained xyloglucan and 1,3,1,4-β-glucans with lower molecular methylation. Based on these results, we concluded that micro- masses resulting from increases in xyloglucan-degrading and gravity affects auxin-related processes at the levels of perception, 1,3,1,4-b-glucanase activities. These results show that increasing signaling, transport and biosynthesis. The data imply that the plant cell wall rigidity via modification of the metabolism of cell effects of microgravity on auxin-related processes observed in wall constituents is an important step in gravity resistance. The previous spaceflight experiments may be related to changes in results shown in Table S5 indicate that numerous genes related to DNA methylation levels. In addition, altered methylation levels of cell wall biosynthesis, such as the gene encoding the cellulose the EIN2 and EIN3 genes, which play important roles in the synthase catalytic subunit, callose synthase genes and cell wall ethylene signaling pathway, were detected. Alterations in the expansion-related genes such as xyloglucan endotransglucosy- methylation of the ethylene responsive transcription factor genes lase, beta-galactosidase and beta-glucosidase, displayed altered ERF11 and ERF094 were also involved in the response to microgravity. Moreover, the methylation levels of the genes methylation levels. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 12 DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. Growth of Arabidopsis seedlings under microgravity conditions not affect gene transcription in Arabidopsis. Compared to genetic variations, modifications in DNA methylation are more The seedlings in the culture chamber installed in cultivation unit flexible. In response to environmental changes, epigenetic two grew for 11 days under microgravity conditions; at the end of modifications enable plants to more easily adapt to new the flight, they were alive and were recovered with the satellite. environments. Although some epigenetic modifications can be We obtained four Arabidopsis seedlings (Col-0) grown in space. reversed, other epigenetic changes are heritable and can be These seedlings were photographed immediately on removal 39,40 passed on at least for one generation. from the satellite and used for the measurement of leaf area. Their In this report, analysis of the whole-genome methylome of first and second true leaves were partially expanded at that time. Arabidopsis seedlings under microgravity conditions revealed We compared the areas of these eight true leaves with those of numerous DMRs. The distribution and methylation levels of the the leaves at the same leaf position of control Arabidopsis DMRs of the five Arabidopsis chromosomes are shown in Figs. seedlings grown on the ground. The average leaf area of the S3–S6. After analysis of the molecular functions of these DMRs, we seedlings grown under microgravity was 0.179 ± 0.023 cm², found that approximately 36 TF genes showed altered methyla- whereas that of the seedlings grown on the ground was tion levels during microgravity spaceflight. It is proposed that the 0.145 ± 0.020 cm². The results show that the leaves of the possible differentially methylated TF genes may contribute to seedlings grown in microgravity were larger than those of the microgravity adaptation in plants (Table S3). Furthermore, ground control samples (Fig. 6). abundant DMRs were found to be involved in “Signaling” pathways, especially in hormone signaling pathways including auxin, ABA, ethylene, and BR (Table S4). Hormone-related genes DISCUSSION whose expression was changed under microgravity conditions Plants are a crucial source of food, oxygen, and pharmaceuticals have also been identified in previous studies of the gene for people and animals, and they are a vital part of human life expression profiles of spaceflight specimens. Here, we were support systems for future long-duration spaceflight. However, particularly concerned with AMR auxin-related genes. In fact, space is not an ideal environment to grow plants, which are alterations in the methylation of auxin-related genes may play affected by microgravity and radiation. Global alterations in gene- important roles in plants’ adaptation to microgravity. Auxin expression patterns have been shown to occur in plants grown in participates in plant gravitropism and is an important hormone 3,4 space. Epigenetic modification of DNA is emerging as an that is involved in the relationship between plants and gravity. A important mechanism for modulating gene expression under previous spaceflight experiment in which GFP signaling was used different environmental conditions. The aim of this study was to to measure the expression of GFP reporter genes in pDR5r::GFP, examine the epigenetic mechanisms that regulate early seedling pTAA1::TAA1–GFP, pSCR::SCR–transgenic Arabidopsis to monitor development in space under microgravity conditions. The results auxin did not detect an influence of microgravity on auxin of this investigation can be used to improve strategies for distribution in the root tip region. However, in other spaceflight designing plant strains that are better able to withstand experiments involving plant cells and seedlings, auxin-related microgravity and other adverse environmental conditions. genes identified by transcriptomic analysis, including genes Epigenetic changes in response to spaceflight lead to altered related to auxin metabolism, transport, perception, and signal cytosine DNA methylation in Arabidopsis seedlings grown under transduction, showed changes in expression in the microgravity microgravity conditions. This research conducted a whole- 2,3 environment. The change in CsPIN1 distribution under micro- genome bisulfate sequencing of Arabidopsis seedlings to discover 30 gravity also affects the local distribution of auxin. Therefore, the and qualitatively assess spaceflight-associated 5-methyl cytosine intensity and distribution of auxin in the cells of different parts of (5mCyt) epigenetic modifications. The results demonstrated that plant organs under microgravity conditions should be further lower methylation levels in the CHG, CHH, and CpG contexts in the characterized using more sensitive approaches. Moreover, signal Arabidopsis genome tended to occur under microgravity condi- transduction by auxin, as well as the mode and intensity of auxin’s tions (Fig. 2). However, this does not mean that all gene patterns action, may be affected by changes in the expression of auxin- were hypomethylated. As the results in Tables S1, S3, and S4 show, related genes under microgravity conditions. Therefore, the methylation-related genes (AGO2, DME, DMT1, and APE1), tran- information on changes in the DNA methylation of auxin-related scription factor genes (MYB65, MYB74, BHLH35, BHLH81, BHLH131, genes presented in this paper contributes to the understanding of BZIP68, GATA26, NAC081, NAC100, NAC105, WRKY7, and WRKY19) the changes in auxin effects and cell wall and plant phenotypes and hormone-related genes (ARF3, IAA9, and IAA33) were all that occur under microgravity conditions. hypermethylated. Some genes, including MYB3R5, GATA14, ARF1, Previous space experiments have shown that altered cell shape ARF2, ARF4, AAO2, BIG, BAK1, ABA, and others, displayed more than occurs as a result of cell wall perturbation under microgravity. two regions with opposite methylation patterns. The actual This phenomenon may also be associated with changes in the changes in the expression of these genes will require further expression of auxin-related genes under microgravity because the experimental verification. Our results cannot be simply interpreted main effect of auxin is to relax the cell wall, thereby increasing cell as indicating that the methylation of DNA is inhibited by growth. Growth induced by auxin is achieved by increasing the microgravity, thereby contributing to hypomethylation of the plasticity of the cell wall. During spaceflight, alterations in the Arabidopsis genome. methylation of cell wall-related genes were identified (Table S5). Previous research on the DNA methylome in Solanum Changes in the expression of this category of genes in lycopersicum, Gossypium hirsutum L., Zea mays, Oryza sativa, and microgravity were also observed in several previous spaceflight 13,34–37 Glycine max provides useful information for analyzing DNA experiments. Fig. 6 shows that the leaf growth of Arabidopsis methylation during Arabidopsis seedling development. Hyper- seedlings was enhanced under microgravity. The most immediate methylation is usually found in centromeres and pericentromeric effect that plants experience under space conditions is weight- regions, and CpG methylation shows the highest levels among the lessness. This is an environment that terrestrial plants have never various species. Our results consistently demonstrated that CHG, encountered. Therefore, the most direct adaptive response of CHH, and CpG methylation patterns were related to TE density. plants is the weakening of mechanical support tissue under These results show that DNA methylation plays an important role microgravity. First, the rigidity of the plant cell wall decreases 4,43 in the regulation of genome stability. In addition, DNA methyla- under microgravity conditions. As a result, the expression of tion has an important function in gene repression, although a cell wall genes is altered. Regulation of these gene-expression recent report indicated that the DNA methylation pattern does changes may be related to changes in the DNA methylation level npj Microgravity (2018) 12 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. in plant cells. We found changes in the DNA methylation levels of MATERIALS AND METHODS genes encoding transcription factors and certain genes associated Experimental setup with cell wall metabolism. Based on these results, the phenotypic To meet the requirements for the SJ-10 spaceflight experiment, surface- acceleration of seedling growth under microgravity may be sterilized seeds of Arabidopsis thaliana (Col-0) were sown in Petri dishes related to changes in the DNA methylome, in the expression of containing half-strength Murashige and Skoog medium and 1% phytagel and stratified at 4 °C for 3 days. After stratification, the Petri dishes auxin-related genes and cell wall-related genes, and in cell wall containing the seeds were placed in a culture room with an ambient rigidity. The results suggest that adjustment of cell wall rigidity by temperature of 22 ± 1 °C and 16 h light at 8000 lx. After germination and modification of the metabolism of the cell wall is an important growth for 3 days in the Petri dishes, 49 seedlings were transferred to mechanism through which plants adapt to changes in gravity. individual culture chambers in cultivation unit 1 and precultivated for These inferences are supported by the results of previous space 3 days until launch (Fig. 1c, cultivation unit 1). The seedlings continued to experiments in which it was demonstrated that increased grow for up to 60 h under microgravity conditions. We fixed these plasticity of the cell wall enhanced the growth of the hypocotyl seedlings with RNAlater® (Ambion, Foster City, CA, USA) under micro- of Arabidopsis, rice coleoptile and flower stems of Arabidopsis gravity at 60 h of spaceflight. After the chemically fixed seedlings were under microgravity conditions. returned to the ground, 49 seedlings in a culture chamber were transferred In addition to methylation alterations at the gene level, TEs are to a Falcon tube containing RNAlater® as a single sample. Three also involved in the adaptation of plants to environmental independent samples prepared in this manner were used for further DNA methylome analysis. The samples were preserved and stored at conditions via epigenetic variations. We demonstrated here that −80 °C before further processing for DNA isolation. The Falcon tubes many TEs display reduced methylation levels under microgravity containing the samples were transferred to −20 °C overnight and then conditions. The relatively unstable DNA methylation of TEs plays thawed at 4 °C overnight. The samples were warmed to room temperature an important role in the induction of active transposons. A to dissolve the RNAlater® precipitate and used for DNA extraction. The global study of TEs under abiotic stress showed that many TEs seedlings, which were grown in cultivation unit 2 under microgravity for show increased expression and that many of them function as 11 days, were still growing when they returned to the ground. All the enhancers of stress-induced genes in the maize genome. In seedlings were maintained under 16 h of light at 8000 lx at 22 ± 1 °C during addition, TE methylation levels may dynamically regulate trans- the spaceflight and on ground. The g-profile during launch of the SJ-10 poson expression in pathogen-infected Arabidopsis. Coupled satellite into orbit is described in the references. The g-profile during with our observations, this indicates that DNA demethylation launch was as follows: the first-level maximum static overload was 4.8 g in within TEs may cause transcriptional alterations in transposons flight for 150 s; the second-level maximum static overload was 6.0 g in during adaptation to spaceflight conditions. Overall, our single- flight for 180 s. base resolution methylome research revealed novel microgravity- induced methylome alterations in Arabidopsis seedlings. These Genome isolation and whole-genome bisulfite sequencing library results are helpful for understanding the role of the regulation of construction methylation in response to microgravity conditions. This protocol was based in part on a previously developed method. Total Because we used Arabidopsis seedlings from the beginning of genomic DNA was isolated from Arabidopsis seedlings according to a the flight experiment, the possible effects from launch conditions previous protocol. Using a sonicator, 5 mg of genomic DNA was on seedlings should also be considered. Therefore, to determine fragmented to a DNA length of approximately 200 bp. The DNA fragments the effects of launch acceleration for 330 s on the DNA were then treated three times using the Qiagen 59104 EpiTect Bisulfite Kit. λ DNA was used to determine the bisulfite conversion rate. Libraries were methylation pattern of Arabidopsis seedlings, we performed a sequenced on the Illumina Hiseq2500 platform. Following library simulated launch experiment on the ground; the results are construction, sequencing of three independent spaceflight samples and shown in Supplementary Fig. S2. We measured changes in DNA three independent ground samples was performed (Fig. S1). methylation levels in specific regions of three genes. As the data show, there were no significant difference in ABA3 and DME, but Mapping of reads some hypermethylation was observed in ARF1 (the methylation Using the default parameters, alignment of bisulfite-treated reads to the tendency of this gene under microgravity was reversed) after Arabidopsis genome was conducted using Bismark software. Sequence hypergravity treatment for 330 s. We, therefore, concluded that reads were also transformed into fully bisulfite-converted versions before exposure to hypergravity for 330 s had little effect on DNA the reads were aligned to similarly converted versions of the genome. methylation in Arabidopsis seedlings that were subsequently Methylation level analysis was conducted by the sliding-window approach, exposed to microgravity for 60 h. Comparison of the results of and all of the unmethylated or methylated reads were counted in the preliminary hypergravity and microgravity experiments showed 50 window. The relative proportions of CHG, CHH, and CpG contexts were that the trend of DNA methylation changes of certain genes in then determined. plants grown under microgravity and hypergravity was reversed. The strength of this correlation should be tested by further DMR analysis extension of the hypergravity processing time and subsequent DMRs were counted using swDMR software with a sliding-window method large-scale analysis. An opposite trend under hypergravity and (http://122.228.158.106/swDMR/). Significantly changed DMRs were microgravity experiments has previously been shown with respect detected by the Fisher test. DMR genes were analyzed. The identified to changes in intracellular calcium concentration. There is also a genes were then analyzed for GO and KEGG (Kyoto Encyclopedia of Genes need for further study, whether there is a relationship between and Genomes) (http://www.genome.jp/kegg/) enrichment. KEGG analysis changes in gravity, changes in intracellular calcium concentration, was used to determine the pathway enrichment of functional DMRs with and changes in DNA methylation. In addition, a 1-g centrifuge was p values less than 0.05. not available due to space limitations within the satellite. To minimize errors, we extracted genomic DNA from one sample Analysis of TEs contained 49 Arabidopsis seedlings, and 3 independent samples TEs were isolated and analyzed using the Repeat Masker program (http:// were used in the analysis. However, we still cannot completely rule www.repeatmasker.org/). We further used Fisher’s exact test to investigate out the possibility that spaceflight disturbances other than TEs with altered methylation levels. The false discovery rate method was microgravity effects affected the results obtained in our space- used for multiple testing adjustments. p Values less than 0.05 was defined flight experiments. as methylated TEs with significant alterations. 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Single-base resolution methylome analysis shows epigenetic changes in Arabidopsis seedlings exposed to microgravity spaceflight conditions on board the SJ-10 recoverable satellite

Xu, Peipei; Chen, Haiying; Jin, Jing; Cai, Weiming
npj Microgravity , Volume 4 (1) – Jul 12, 2018
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Life Sciences; Life Sciences, general; Classical and Continuum Physics; Biotechnology; Immunology; Space Sciences (including Extraterrestrial Physics, Space Exploration and Astronautics) ; Applied Microbiology
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www.nature.com/npjmgrav ARTICLE OPEN Single-base resolution methylome analysis shows epigenetic changes in Arabidopsis seedlings exposed to microgravity spaceflight conditions on board the SJ-10 recoverable satellite 1 1 1 1 Peipei Xu , Haiying Chen , Jing Jin and Weiming Cai DNA methylation is a very important epigenetic modification that participates in many biological functions. Although many studies of DNA methylation have been reported in various plant species, few studies have assessed the global DNA methylation pattern in plants challenged by exposure to microgravity conditions. In this report, we mapped the Arabidopsis genome methylation pattern changes associated with microgravity conditions on board the Chinese recoverable scientific satellite SJ-10 at single-base resolution. Interestingly, we found epigenetic differences in Arabidopsis seedlings exposed to microgravity in that the Arabidopsis genome exhibits lower methylation levels in the CHG, CHH, and CpG contexts under microgravity conditions. Microgravity stimulation was related to altered methylation of a number of genes, including DNA methylation-associated genes, hormone signaling related genes, cell-wall modification genes and transposable elements (TEs). Relatively unstable DNA methylation of TEs was responsible for the induction of active transposons. These observations suggest that DNA demethylation within TEs may affect the transcription of transposons in response to microgravity conditions. In summary, the results of this investigation are beneficial for understanding the mechanism of plant adaptation to microgravity and improve strategies to allow plants to adapt to space. npj Microgravity (2018) 4:12 ; doi:10.1038/s41526-018-0046-z INTRODUCTION indicate an association between environmental stresses and DNA methylation alterations. On Earth, life is adapted to a constant gravitational force, and DNA cytosine methylation functions as an important controller biological processes in organisms have evolved under this natural of gene mRNA levels. In the Arabidopsis genome, DNA cytosine constant. Many limiting factors for plant growth under micro- methylation is mostly detected at CpG sites and can also occur at gravity conditions have been identified. Therefore, it is expected CHG and CHH residues (H indicates A, T, or C). In the Arabidopsis that plants have sufficient and sustainable mechanisms that allow genome, most methylation in the gene body is detected at CpG for them to survive under microgravity conditions. To identify sites, while CpG, CHH, and CHG site methylation occurs elsewhere plant responses to the microgravity stimulus, many groups have 12 and in repetitive regions. CG methylation is enriched in TEs and been using various approaches to identify genes with altered 13 gene bodies, but non-CG methylation is mostly present in TEs. 1–5 expression levels under microgravity conditions. The results Often, methylation of gene promoter regions is believed to inhibit have shown that microgravity signals can be transduced into gene expression, while methylation within introns and exons can molecular signaling cascades, which lead to plant adaptation. promote gene transcription. MET1 is the main CG methyltrans- Currently, it is obvious that epigenetic alterations are involved ferase, CMT3 is the main CHG methyltransferase, and DRM2 is the in plant adaptation to environmental stress. A series of current main CHH methyltransferase and is guided by small RNAs. reports have shown that DNA methylation leads to control of gene Epigenetic features play a critical role in controlling gene mRNA levels and plays important roles in abiotic and biotic expression and the subsequent response of an organism to its stresses. For example, drought stress results in a global DNA environment. As a major epigenetic modification, DNA methylation is not directly encoded in the genome sequence, and yet can methylation alteration in rice. Salt stress induces OsMYB91 gene modify expression and may be inherited for at least one expression due to the reduced cytosine methylation level in its generation. Several studies have shown that large numbers of promoter region. In addition, widespread alterations in DNA plant genes are differentially expressed in response to spaceflight. methylation of the poplar genome in response to drought 9 Learning more about the spaceflight methylome of plants will treatment indicate adaption to the local environment. Moreover, contribute to the foundational understanding of how plants adapt epigenetic variations in transposable element (TE) regions can be 10 to spaceflight. In this research, we showed the results of an SJ-10 affected by environmental stresses. Silencing of the ZmMET1 spaceflight experiment. The experiment was a part of the SJ-10 gene after low-temperature treatment leads to demethylation of mission, an experimental project of Chinese Academy of Sciences the Ac/Ds transposon region in maize roots. In sum, these results in April 2016. Arabidopsis seedlings were exposed to spaceflight on Laboratory of Photosynthesis and Environment, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, No. 300 Fenglin Road, Shanghai 200032, China Correspondence: Weiming Cai (wmcai@sibs.ac.cn) These authors contributed equally: Peipei Xu, Haiying Chen. Received: 14 December 2017 Revised: 28 May 2018 Accepted: 1 June 2018 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. board of SJ-10. To evaluate DNA methylome during spaceflight, we other histogram tracks represent differentially methylated regions profiled DNA methylation genome wide in Arabidopsis seedlings. (DMRs) in the CHG (d), CHH (e), and CpG (f) contexts. Whole-genome bisulfite sequencing (BS-Seq) allows DNA methyla- Previous research showing that cytosine methyltransferases and tion to be measured at the whole-genome level with single demethylases play important roles in the maintenance of genomic nucleotide resolution. These results will be very useful for our methylation indicates that these enzymes are involved in a understanding of the potential role of cytosine DNA methylation in number of biological functions. To investigate possible altered plants adapting to the outer space environment. methylation patterns in methyltransferase- and demethylase- related genes in response to microgravity conditions, we examined all of these genes to determine whether there were RESULTS any changes in their DNA methylation levels under microgravity. Experimental design and sample fixation on the recoverable The details of these results for DMT1, which encodes the cytosine scientific satellite SJ-10 methyltransferase MET1, DME, which encodes the DNA glycosy- lase DEMETER, and TET14, a tetraspanin family gene, are shown in The specific equipment used in the space experiment was Table S1. The results indicate that exposure of the plants to designed and constructed by Shanghai Institution of Technical microgravity was accompanied by alterations in the DNA Physics (Fig. 1a, b). The equipment included two cultivation units, methylation patterns of specific genes. a canal system and a pump support system. The polysulfone chambers in the cultivation units had windows that were covered by a gas permeable membrane. The chambers were connected to Altered methylation profiles of functional genes in response to the fixative unit by tube connectors. The Chinese recoverable microgravity conditions scientific satellite SJ-10 was launched at 01:38 on April 6, 2016 and To perform an in-depth analysis of the functional categories of landed at 16:30 on April 18, 2016. The air temperature near the genes that showed alterations in their methylation patterns under incubator averaged approximately 23 °C. The average relative microgravity conditions, we used gene ontology (GO) enrichment humidity near the incubator was approximately 25%, and the to categorize these genes. In the CHG context in the category of −3 microgravity level was estimated to be approximately 10 to biological processes, altered methylation-related (AMR) genes −4 15,16 10 g during the unified flight phase. Seedlings were fixed were enriched for cell wall-related processes, carbohydrate with RNAlater® after growth for 60 h under microgravity condi- metabolic processes, defense response, and nitrogen compound tions. They were harvested approximately 4 h after landing, stored transport. Changes in these biological processes were also shown at 4 °C, and transported to our laboratory for further assays. in transcriptomic studies in several previous plant spaceflight Seedling DNA was extracted and used for methylome analysis. To 3,4 experiments. Although the results of various spaceflight halt cellular activities in space and preserve the DNA methylome experiments differ for a variety of reasons, changes in cell wall- profile, RNAlater® was added to the culture chambers on board SJ- related processes and defense response were consistently 10 and on the ground. RNAlater® has been experimentally verified 4 detected. This suggests that the weakening of the plant cell wall to effectively preserve DNA and to yield high-quality RNA and has that occurs under microgravity may be regulated at the level of been used successfully in spaceflight applications. Samples of DNA methylation. Changes in metabolism-associated AMR genes, the 1 g ground controls in cultivation unit 1 were also fixed in including carbohydrate metabolic processes and nitrogen com- RNAlater® at the same time as the in-space samples (Fig. 1d). pound transport, suggested that energy metabolism might be regulated in response to the microgravity environment. The Global DNA methylation patterns of Arabidopsis seedlings under defense response that occurs in plants during spaceflight might microgravity conditions be due to the changes in forces on the cell wall under microgravity. The discovery that defense response AMR genes To identify the DNA methylation pattern that was present in the are expressed under microgravity may validate this assumption seedlings during spaceflight, single-base resolution methylome (Fig. 3a). In the CHH context in the category of biological analysis was conducted using Arabidopsis (Col-0) seedlings that processes, AMR genes for metabolic processes, cellular metabolic had been grown for 60 h under microgravity conditions. The DNA processes, biosynthetic processes, signaling transduction, methylation profiles at the whole-genome level were determined; oxidation–reduction processes, and lipid biosynthesis were analysis of the read intensity around the annotated genes enriched. Lipid biosynthesis has been implicated in gravity revealed lower DNA methylation levels under microgravity conditions (Fig. 2). Because euchromatin and heterochromatic responses in Arabidopsis, and is likely involved in adjusting the genomic regions such as coding sequences and repetitive polarity of cells. Here, we also found the lipid biosynthesis AMR sequence regions display different DNA methylation patterns, genes. Gravity sensing in plant cells involves signals related to we further evaluated the detailed methylation patterns within oxidative stress. In fact, oxidation–reduction process AMR genes genes, including coding sequences (CDS) and noncoding areas. were also identified. Regarding cellular components such as cell, The results showed that CpG sites had the highest methylation intracellular, organelle, plasma membrane, and cell periphery, levels and that CHH sites had the lowest methylation levels in AMR genes were enriched (Fig. 3b). In the CpG context in the each gene region (Fig. 2a–c). In the CpG context, CpG methylation category of biological processes, AMR genes for mRNA transport, in the 5’UTR and 3’UTR regions was lower than that in coding mRNA export from the nucleus, microtubule-based movement, regions, and the 5’UTR methylation level was much lower than telomere maintenance in response to DNA damage, regulation of that in the 1500-bp upstream region and in the 3’UTR (Fig. 2c). The telomere maintenance and inositol phosphate phosphorylation CHG context appeared to be quite different from the CpG context, were enriched. Because space radiation can cause damage to DNA with much higher methylation levels in 1500-bp upstream regions directly, the radio-adaptive response (RAR) of Arabidopsis root and 5’UTR regions, and CDS displayed the lowest methylation growth is modulated under microgravity conditions, and DNA level (Fig. 2a). However, the CHH context showed higher damage repair in RAR is regulated by microgravity. Here, we methylation levels in the 5’UTR and 3’UTR regions and lower found DNA damage and regulation of telomere maintenance AMR methylation levels in the 1500-bp upstream region and CDS sites genes, supporting this assumption. Inositol phosphate signaling (Fig. 2b). Circos plots show the differences in methylation changes has also been shown to occur in response to microgravity between ground controls (G1–G3) and plants exposed to conditions. For cellular components, AMR genes for the microgravity conditions (S1–S3) (Fig. 2d–f). The outer track functional categories of Cul4-RING ubiquitin ligase complex, indicates the five chromosomes of the Arabidopsis genome. The nuclear envelope, and nuclear pore were enriched (Fig. 3c). npj Microgravity (2018) 12 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. Fig. 1 Hardware and experimental procedures used in the experiment on board the SJ-10 recoverable satellite. a Appearance of the hardware used in the spaceflight experiment. The equipment functioned as a housing for the culture chambers. b Distribution of internal components of the hardware. The hardware includes culture chambers, a pump support system, a canal system and a fixative unit connected to the culture chambers. c Arabidopsis seedlings were grown in culture chambers during the space experiment. d Schematic view of the Arabidopsis seedlings on board the SJ-10 satellite during spaceflight and on the ground. Arabidopsis seedlings in culture chambers were transferred from Petri dishes 3 days before launching. Seedlings in culture chambers installed in cultivation unit 1 were grown for 60 h under microgravity conditions and fixed in space using RNAlater®. The seedlings in the culture chamber installed in cultivation unit two grew for 11 days under microgravity; after this time, they were still alive and returned with the satellite. The g-profile during SJ-10 satellite in orbit is described in the 15,16 references. The g-profile during launch was as follows: the first-level maximum static overload was 4.8 g in flight for 150 s; the second-level maximum static overload was 6.0 g in flight for 180 s Numerous TE genes are hypomethylated under microgravity CpG contexts in the TE regions and methylation levels in the 1-kb conditions upstream and downstream regions were demonstrated (Fig. 4a). TEs presented lower methylation levels in the CHG, CHH, and CpG Previous research has shown that mobilization and silencing of contexts in spaceflight samples. The results showed that a large TEs are often accompanied by disruption of DNA methylation. TEs can affect the size of the genome, create insertions and other proportion of the TEs in these contexts are differentially mutations, and influence gene expression patterns. Under methylated and that hypomethylation of TE sites was likely the microgravity conditions, many differentially methylated TEs were result of the induction of active TEs. demethylated (Fig. 4a). The methylation patterns of the CHG, CHH, Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 12 DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. Fig. 2 DNA methylation patterns in various genomic regions. a–c Distribution of the DNA methylation levels in the CHG a, CHH b, and CpG c contexts among various gene regions, including the promoter, 5’UTR, gene body and 3’UTR in ground controls (G1–G3), and in plants maintained under microgravity conditions (S1–S3). d–f Circos plots showing Arabidopsis genomic methylation in ground controls (G1–G3) and in plants maintained under microgravity conditions (S1–S3). The outer track represents five chromosomes of the Arabidopsis genome. The other histogram tracks represent differentially methylated regions in the CHG d, CHH e, and CpG f contexts Altered methylation profiles of TEs of different lengths in response whereas in the CHH context they were assigned to pathways to microgravity conditions related to metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and ribosomes (Fig. Previous research has shown that the length of TEs can affect their 5b). In the CpG context, they were mainly assigned to pathways methylation levels. Moreover, TE methylation levels can associated with metabolism-related processes (such as sulfur influence transposon expression in response to environmental metabolism, terpenoid backbone metabolism, N-glycan biosynth- stress. To further identify the association between methylation esis, butanoate metabolism, and beta-alanine metabolism), ABC status and TE length in Arabidopsis, TEs were divided into three transporters, nucleotide excision repair, protein procession in the groups: the first group with lengths shorter than 500 bp, second ER, and endocytosis (Fig. 5c). Many of the pathways identified here group with lengths ranging from 500 to 2000 bp; and third group in the studies of transcriptomic analysis under microgravity have with lengths of more than 2000 bp. Compared with the ground 3,4 also been found in other spaceflight experiments. Some of controls (G1–G3), the first group of TEs showed a striking decrease these pathways play important roles in plant gravity perception in CHG and CHH methylation levels under microgravity conditions and in the responses of plants to microgravity. For instance, ABC (S1–S3) (Fig. 4b). The second group showed a marginal decrease transporters have been shown to play a role in the transport of the in the CHG and CHH methylation contexts (Fig. 4c). The third phytohormone auxin. The enrichment of AMR genes encoding group showed a decrease in the CHG context but no decrease in ABC transporters implies that auxin transport can be modulated the CHH or CpG methylation contexts under microgravity by microgravity conditions at the level of DNA methylation. conditions (Fig. 4d). We propose that demethylation of TEs plays We also further characterized methylation alterations in genes an important role under microgravity conditions. encoding transcription factors (TFs). The PlantTFDB predicts that the Arabidopsis genome contains more than 2200 TFs. Approxi- Widespread DNA methylation in response to microgravity mately, 36 TF genes showed altered methylation levels during spaceflight microgravity spaceflight. These included bHLH, bZIP, GATA, WRKY, To identify the potential influence of microgravity spaceflight on and NAC members (Table S3). Previous spaceflight experiments the methylation level of the whole genome, we analyzed the demonstrated that the expression of many TF genes is altered 2,3 DMRs in flight seedlings and ground controls. The distribution and under microgravity. For example, in the TROPI-2 project, an NAC methylation levels of the DMRs of five Arabidopsis chromosomes TF gene, ANAC104/XND1 (At5g64530), was up regulated under are shown in Figs. S3–S6. To determine the molecular functions of microgravity conditions. ANAC104/XND1 regulates the synthesis these DMR-related genes, we conducted KEGG pathway enrich- of tracheal element secondary walls and can affect plant height ment analysis. In the CHG context, these genes were mainly and the length of tracheal elements in stems and hypocotyls. assigned to pathways associated with metabolism (starch and Another NAC TF gene, NAC1 (NAC domain-containing protein 21/ sucrose metabolism, phenylalanine biosynthesis, purine metabo- 22, At1g56010), was down regulated under microgravity. NAC1 lism, glutathione metabolism, amino sugar, and nucleotide sugar encodes a transcription factor that is induced by auxin and metabolism), proteasomes, ribosomes, and phagosomes (Fig. 5a), mediates auxin signaling to promote lateral root development. npj Microgravity (2018) 12 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. Fig. 3 GO-term enrichment network of genes with altered methylation levels in the Arabidopsis genome. GO-term enrichment analysis of altered gene methylation patterns in the CHG a, CHH b and CpG c contexts under microgravity conditions was conducted. The bar represents the number of genes in the test set belonging to each GO category. Only the top 10 GO terms are listed (p < 0.05) Alteration of the expression of auxin-related and cell wall-related under microgravity have been identified, including the genes genes under microgravity is closely related to plant adaptation to encoding auxin responsive factors (ARF1-6, ARF8, ARF19, ARF21, microgravity. Therefore, it is proposed that the possible differential and ARF22), auxin signaling F-box proteins (TIR1, AFB2, and AFB5), methylation of genes encoding TFs may also contribute to the auxin responsive proteins (IAA9 and IAA33), transport protein (BIG) adaptation of plants to microgravity. and indole-3-acetaldehyde oxidases (AAO1 and AAO2). TIR1 Abundant DMRs were found to be involved in the “Signaling” encodes an auxin receptor that mediates auxin-regulated pathway. The observed alterations in the methylation of genes transcription. The observation of altered TIR1 methylation levels involved in hormone signaling pathways were functionally implies that microgravity affects auxin signaling in plants. Aux/ characterized. Genes involved in hormone signaling pathways, IAA proteins are short-lived TFs that function as repressors of early including auxin, ABA (abscisic acid), ethylene, and BRs (brassinos- auxin response genes. Repression is thought to result from the teroids), were also differentially methylated (Table S4). Notably, interaction of the repressors with auxin response factors (ARFs). several auxin-related genes whose DNA methylation was changed Formation of heterodimers with ARF proteins may alter the ability Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 12 DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. Fig. 4 Methylation patterns of TEs and different types of TEs with different lengths in the Arabidopsis genome. a Methylation patterns of TEs. The distribution of the methylation read density for all TEs in the Arabidopsis genome between ground controls (G1–G3), and under microgravity conditions (S1–S3) is shown. The x-axis represents the TE body and its 1-kb upstream and downstream regions. The y-axis indicates the average methylation level. Three types of methylation patterns, CHG, CHH, and CpG, are shown in the figure. b–d Methylation patterns of TEs with different lengths. Methylation patterns of TEs with different lengths in ground controls (G1–G3) and in plants grown under microgravity conditions (S1–S3). TEs were divided into quintiles based on their lengths: the first quintile is the shortest, and the third quintile represents TEs of the longest length. The read density distribution for TEs with lengths of (b) less than 500 bp, (c) 500–2000 bp, and (d) more than 2000 bp within the Arabidopsis genome is shown. Three types of methylation patterns, CHG, CHH, and CpG, are shown in each figure npj Microgravity (2018) 12 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. Fig. 5 Pathway analysis of genes that showed altered methylation. KEGG pathway enrichment analysis of CHG a, CHH b, and CpG c differentially methylated genes in the Arabidopsis genome in plants grown under microgravity conditions and ground controls was performed. The size of the circle represents the gene numbers, and the color represents the q value Fig. 6 Comparison of the phenotypes of Arabidopsis seedlings grown under microgravity and on the ground. a Seedlings that served as ground control samples. b Seedlings that returned to earth with the satellite after growth under microgravity for 11 days. The experimental procedure was the same as described in Fig. 1d. The leaf area was measured using ImageJ. Bar = 1 cm (in a, b). The data shown are the mean values obtained for eight leaves (n = 8); the vertical bars represent the standard deviation. Asterisks indicate a statistically significant difference (p < 0.05) between spaceflight and control samples using Student’s two-tailed t test of these proteins to modulate the expression of genes involved in involved in ABA biosynthesis (ABA3) and brassinosteroid insensi- the early auxin response. The decreased methylation level of ARF tive1 associated receptor kinase (BAK1) were also decreased. genes and the increased methylation level of the IAA9 and IAA33 Alterations in the methylation of genes related to cell wall were genes may contribute to an increased auxin response. The also identified. Hypergravity has been shown to upregulate cell gravimorphogenesis of plant organs was changed due to the wall rigidity in stems and roots. However, plant cell wall rigidity effects of microgravity on the polar transport of auxin. BIG is lower under microgravity conditions than under ground encodes a membrane-associated protein and is required for auxin conditions. The thickness of cell walls decreased in response transport. We found that BIG was hypermethylated in space; thus, to the microgravity stimulus. The space-grown plants also microgravity might inhibit auxin transport by increasing BIG contained xyloglucan and 1,3,1,4-β-glucans with lower molecular methylation. Based on these results, we concluded that micro- masses resulting from increases in xyloglucan-degrading and gravity affects auxin-related processes at the levels of perception, 1,3,1,4-b-glucanase activities. These results show that increasing signaling, transport and biosynthesis. The data imply that the plant cell wall rigidity via modification of the metabolism of cell effects of microgravity on auxin-related processes observed in wall constituents is an important step in gravity resistance. The previous spaceflight experiments may be related to changes in results shown in Table S5 indicate that numerous genes related to DNA methylation levels. In addition, altered methylation levels of cell wall biosynthesis, such as the gene encoding the cellulose the EIN2 and EIN3 genes, which play important roles in the synthase catalytic subunit, callose synthase genes and cell wall ethylene signaling pathway, were detected. Alterations in the expansion-related genes such as xyloglucan endotransglucosy- methylation of the ethylene responsive transcription factor genes lase, beta-galactosidase and beta-glucosidase, displayed altered ERF11 and ERF094 were also involved in the response to microgravity. Moreover, the methylation levels of the genes methylation levels. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 12 DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. Growth of Arabidopsis seedlings under microgravity conditions not affect gene transcription in Arabidopsis. Compared to genetic variations, modifications in DNA methylation are more The seedlings in the culture chamber installed in cultivation unit flexible. In response to environmental changes, epigenetic two grew for 11 days under microgravity conditions; at the end of modifications enable plants to more easily adapt to new the flight, they were alive and were recovered with the satellite. environments. Although some epigenetic modifications can be We obtained four Arabidopsis seedlings (Col-0) grown in space. reversed, other epigenetic changes are heritable and can be These seedlings were photographed immediately on removal 39,40 passed on at least for one generation. from the satellite and used for the measurement of leaf area. Their In this report, analysis of the whole-genome methylome of first and second true leaves were partially expanded at that time. Arabidopsis seedlings under microgravity conditions revealed We compared the areas of these eight true leaves with those of numerous DMRs. The distribution and methylation levels of the the leaves at the same leaf position of control Arabidopsis DMRs of the five Arabidopsis chromosomes are shown in Figs. seedlings grown on the ground. The average leaf area of the S3–S6. After analysis of the molecular functions of these DMRs, we seedlings grown under microgravity was 0.179 ± 0.023 cm², found that approximately 36 TF genes showed altered methyla- whereas that of the seedlings grown on the ground was tion levels during microgravity spaceflight. It is proposed that the 0.145 ± 0.020 cm². The results show that the leaves of the possible differentially methylated TF genes may contribute to seedlings grown in microgravity were larger than those of the microgravity adaptation in plants (Table S3). Furthermore, ground control samples (Fig. 6). abundant DMRs were found to be involved in “Signaling” pathways, especially in hormone signaling pathways including auxin, ABA, ethylene, and BR (Table S4). Hormone-related genes DISCUSSION whose expression was changed under microgravity conditions Plants are a crucial source of food, oxygen, and pharmaceuticals have also been identified in previous studies of the gene for people and animals, and they are a vital part of human life expression profiles of spaceflight specimens. Here, we were support systems for future long-duration spaceflight. However, particularly concerned with AMR auxin-related genes. In fact, space is not an ideal environment to grow plants, which are alterations in the methylation of auxin-related genes may play affected by microgravity and radiation. Global alterations in gene- important roles in plants’ adaptation to microgravity. Auxin expression patterns have been shown to occur in plants grown in participates in plant gravitropism and is an important hormone 3,4 space. Epigenetic modification of DNA is emerging as an that is involved in the relationship between plants and gravity. A important mechanism for modulating gene expression under previous spaceflight experiment in which GFP signaling was used different environmental conditions. The aim of this study was to to measure the expression of GFP reporter genes in pDR5r::GFP, examine the epigenetic mechanisms that regulate early seedling pTAA1::TAA1–GFP, pSCR::SCR–transgenic Arabidopsis to monitor development in space under microgravity conditions. The results auxin did not detect an influence of microgravity on auxin of this investigation can be used to improve strategies for distribution in the root tip region. However, in other spaceflight designing plant strains that are better able to withstand experiments involving plant cells and seedlings, auxin-related microgravity and other adverse environmental conditions. genes identified by transcriptomic analysis, including genes Epigenetic changes in response to spaceflight lead to altered related to auxin metabolism, transport, perception, and signal cytosine DNA methylation in Arabidopsis seedlings grown under transduction, showed changes in expression in the microgravity microgravity conditions. This research conducted a whole- 2,3 environment. The change in CsPIN1 distribution under micro- genome bisulfate sequencing of Arabidopsis seedlings to discover 30 gravity also affects the local distribution of auxin. Therefore, the and qualitatively assess spaceflight-associated 5-methyl cytosine intensity and distribution of auxin in the cells of different parts of (5mCyt) epigenetic modifications. The results demonstrated that plant organs under microgravity conditions should be further lower methylation levels in the CHG, CHH, and CpG contexts in the characterized using more sensitive approaches. Moreover, signal Arabidopsis genome tended to occur under microgravity condi- transduction by auxin, as well as the mode and intensity of auxin’s tions (Fig. 2). However, this does not mean that all gene patterns action, may be affected by changes in the expression of auxin- were hypomethylated. As the results in Tables S1, S3, and S4 show, related genes under microgravity conditions. Therefore, the methylation-related genes (AGO2, DME, DMT1, and APE1), tran- information on changes in the DNA methylation of auxin-related scription factor genes (MYB65, MYB74, BHLH35, BHLH81, BHLH131, genes presented in this paper contributes to the understanding of BZIP68, GATA26, NAC081, NAC100, NAC105, WRKY7, and WRKY19) the changes in auxin effects and cell wall and plant phenotypes and hormone-related genes (ARF3, IAA9, and IAA33) were all that occur under microgravity conditions. hypermethylated. Some genes, including MYB3R5, GATA14, ARF1, Previous space experiments have shown that altered cell shape ARF2, ARF4, AAO2, BIG, BAK1, ABA, and others, displayed more than occurs as a result of cell wall perturbation under microgravity. two regions with opposite methylation patterns. The actual This phenomenon may also be associated with changes in the changes in the expression of these genes will require further expression of auxin-related genes under microgravity because the experimental verification. Our results cannot be simply interpreted main effect of auxin is to relax the cell wall, thereby increasing cell as indicating that the methylation of DNA is inhibited by growth. Growth induced by auxin is achieved by increasing the microgravity, thereby contributing to hypomethylation of the plasticity of the cell wall. During spaceflight, alterations in the Arabidopsis genome. methylation of cell wall-related genes were identified (Table S5). Previous research on the DNA methylome in Solanum Changes in the expression of this category of genes in lycopersicum, Gossypium hirsutum L., Zea mays, Oryza sativa, and microgravity were also observed in several previous spaceflight 13,34–37 Glycine max provides useful information for analyzing DNA experiments. Fig. 6 shows that the leaf growth of Arabidopsis methylation during Arabidopsis seedling development. Hyper- seedlings was enhanced under microgravity. The most immediate methylation is usually found in centromeres and pericentromeric effect that plants experience under space conditions is weight- regions, and CpG methylation shows the highest levels among the lessness. This is an environment that terrestrial plants have never various species. Our results consistently demonstrated that CHG, encountered. Therefore, the most direct adaptive response of CHH, and CpG methylation patterns were related to TE density. plants is the weakening of mechanical support tissue under These results show that DNA methylation plays an important role microgravity. First, the rigidity of the plant cell wall decreases 4,43 in the regulation of genome stability. In addition, DNA methyla- under microgravity conditions. As a result, the expression of tion has an important function in gene repression, although a cell wall genes is altered. Regulation of these gene-expression recent report indicated that the DNA methylation pattern does changes may be related to changes in the DNA methylation level npj Microgravity (2018) 12 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA DNA Methylome Analysis in Arabidopsis under Microgravity P Xu et al. in plant cells. We found changes in the DNA methylation levels of MATERIALS AND METHODS genes encoding transcription factors and certain genes associated Experimental setup with cell wall metabolism. Based on these results, the phenotypic To meet the requirements for the SJ-10 spaceflight experiment, surface- acceleration of seedling growth under microgravity may be sterilized seeds of Arabidopsis thaliana (Col-0) were sown in Petri dishes related to changes in the DNA methylome, in the expression of containing half-strength Murashige and Skoog medium and 1% phytagel and stratified at 4 °C for 3 days. After stratification, the Petri dishes auxin-related genes and cell wall-related genes, and in cell wall containing the seeds were placed in a culture room with an ambient rigidity. The results suggest that adjustment of cell wall rigidity by temperature of 22 ± 1 °C and 16 h light at 8000 lx. After germination and modification of the metabolism of the cell wall is an important growth for 3 days in the Petri dishes, 49 seedlings were transferred to mechanism through which plants adapt to changes in gravity. individual culture chambers in cultivation unit 1 and precultivated for These inferences are supported by the results of previous space 3 days until launch (Fig. 1c, cultivation unit 1). The seedlings continued to experiments in which it was demonstrated that increased grow for up to 60 h under microgravity conditions. We fixed these plasticity of the cell wall enhanced the growth of the hypocotyl seedlings with RNAlater® (Ambion, Foster City, CA, USA) under micro- of Arabidopsis, rice coleoptile and flower stems of Arabidopsis gravity at 60 h of spaceflight. After the chemically fixed seedlings were under microgravity conditions. returned to the ground, 49 seedlings in a culture chamber were transferred In addition to methylation alterations at the gene level, TEs are to a Falcon tube containing RNAlater® as a single sample. Three also involved in the adaptation of plants to environmental independent samples prepared in this manner were used for further DNA methylome analysis. The samples were preserved and stored at conditions via epigenetic variations. We demonstrated here that −80 °C before further processing for DNA isolation. The Falcon tubes many TEs display reduced methylation levels under microgravity containing the samples were transferred to −20 °C overnight and then conditions. The relatively unstable DNA methylation of TEs plays thawed at 4 °C overnight. The samples were warmed to room temperature an important role in the induction of active transposons. A to dissolve the RNAlater® precipitate and used for DNA extraction. The global study of TEs under abiotic stress showed that many TEs seedlings, which were grown in cultivation unit 2 under microgravity for show increased expression and that many of them function as 11 days, were still growing when they returned to the ground. All the enhancers of stress-induced genes in the maize genome. In seedlings were maintained under 16 h of light at 8000 lx at 22 ± 1 °C during addition, TE methylation levels may dynamically regulate trans- the spaceflight and on ground. The g-profile during launch of the SJ-10 poson expression in pathogen-infected Arabidopsis. Coupled satellite into orbit is described in the references. The g-profile during with our observations, this indicates that DNA demethylation launch was as follows: the first-level maximum static overload was 4.8 g in within TEs may cause transcriptional alterations in transposons flight for 150 s; the second-level maximum static overload was 6.0 g in during adaptation to spaceflight conditions. Overall, our single- flight for 180 s. base resolution methylome research revealed novel microgravity- induced methylome alterations in Arabidopsis seedlings. These Genome isolation and whole-genome bisulfite sequencing library results are helpful for understanding the role of the regulation of construction methylation in response to microgravity conditions. This protocol was based in part on a previously developed method. Total Because we used Arabidopsis seedlings from the beginning of genomic DNA was isolated from Arabidopsis seedlings according to a the flight experiment, the possible effects from launch conditions previous protocol. Using a sonicator, 5 mg of genomic DNA was on seedlings should also be considered. Therefore, to determine fragmented to a DNA length of approximately 200 bp. The DNA fragments the effects of launch acceleration for 330 s on the DNA were then treated three times using the Qiagen 59104 EpiTect Bisulfite Kit. λ DNA was used to determine the bisulfite conversion rate. Libraries were methylation pattern of Arabidopsis seedlings, we performed a sequenced on the Illumina Hiseq2500 platform. Following library simulated launch experiment on the ground; the results are construction, sequencing of three independent spaceflight samples and shown in Supplementary Fig. S2. We measured changes in DNA three independent ground samples was performed (Fig. S1). methylation levels in specific regions of three genes. As the data show, there were no significant difference in ABA3 and DME, but Mapping of reads some hypermethylation was observed in ARF1 (the methylation Using the default parameters, alignment of bisulfite-treated reads to the tendency of this gene under microgravity was reversed) after Arabidopsis genome was conducted using Bismark software. Sequence hypergravity treatment for 330 s. We, therefore, concluded that reads were also transformed into fully bisulfite-converted versions before exposure to hypergravity for 330 s had little effect on DNA the reads were aligned to similarly converted versions of the genome. methylation in Arabidopsis seedlings that were subsequently Methylation level analysis was conducted by the sliding-window approach, exposed to microgravity for 60 h. Comparison of the results of and all of the unmethylated or methylated reads were counted in the preliminary hypergravity and microgravity experiments showed 50 window. The relative proportions of CHG, CHH, and CpG contexts were that the trend of DNA methylation changes of certain genes in then determined. plants grown under microgravity and hypergravity was reversed. The strength of this correlation should be tested by further DMR analysis extension of the hypergravity processing time and subsequent DMRs were counted using swDMR software with a sliding-window method large-scale analysis. An opposite trend under hypergravity and (http://122.228.158.106/swDMR/). Significantly changed DMRs were microgravity experiments has previously been shown with respect detected by the Fisher test. DMR genes were analyzed. The identified to changes in intracellular calcium concentration. There is also a genes were then analyzed for GO and KEGG (Kyoto Encyclopedia of Genes need for further study, whether there is a relationship between and Genomes) (http://www.genome.jp/kegg/) enrichment. KEGG analysis changes in gravity, changes in intracellular calcium concentration, was used to determine the pathway enrichment of functional DMRs with and changes in DNA methylation. In addition, a 1-g centrifuge was p values less than 0.05. not available due to space limitations within the satellite. To minimize errors, we extracted genomic DNA from one sample Analysis of TEs contained 49 Arabidopsis seedlings, and 3 independent samples TEs were isolated and analyzed using the Repeat Masker program (http:// were used in the analysis. However, we still cannot completely rule www.repeatmasker.org/). We further used Fisher’s exact test to investigate out the possibility that spaceflight disturbances other than TEs with altered methylation levels. The false discovery rate method was microgravity effects affected the results obtained in our space- used for multiple testing adjustments. p Values less than 0.05 was defined flight experiments. as methylated TEs with significant alterations. 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