Abstract

   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.

Plant biology: Spaceflight leaves its epigenetic mark on seedling DNA

   Spaceflight alters the pattern of chemical tags that adorn DNA in plant
   seedlings. Weiming Cai and colleagues from the Shanghai Institute of
   Plant Physiology and Ecology, China, profiled the genome-wide
   epigenetic patterns of Arabidopsis thaliana seedlings that spent 60 h
   in the microgravity of low Earth orbit aboard the Shijian-10
   recoverable satellite. They analyzed the distribution of methyl tags
   across the genome — an epigenetic mark that affects gene expression
   levels — and found that seedlings exposed to microgravity had lower
   methylation on average than control plants grown on the ground,
   although certain genes related to methylation, transcription factors
   and hormones tended to be more methylated. Epigenetic differences were
   also observed among genes involved in cell-wall modification and in
   transposable elements. The findings could help inform the design of
   plants optimized for growth in space.

Introduction

   On Earth, life is adapted to a constant gravitational force, and
   biological processes in organisms have evolved under this natural
   constant. Many limiting factors for plant growth under microgravity
   conditions have been identified. Therefore, it is expected that plants
   have sufficient and sustainable mechanisms that allow for them to
   survive under microgravity conditions. To identify plant responses to
   the microgravity stimulus, many groups have been using various
   approaches to identify genes with altered expression levels under
   microgravity conditions.^[30]1–[31]5 The results have shown that
   microgravity signals can be transduced into molecular signaling
   cascades, which lead to plant adaptation.

   Currently, it is obvious that epigenetic alterations are involved in
   plant adaptation to environmental stress.^[32]6 A series of current
   reports have shown that DNA methylation leads to control of gene mRNA
   levels and plays important roles in abiotic and biotic stresses.^[33]6
   For example, drought stress results in a global DNA methylation
   alteration in rice.^[34]7 Salt stress induces OsMYB91 gene expression
   due to the reduced cytosine methylation level in its promoter
   region.^[35]8 In addition, widespread alterations in DNA methylation of
   the poplar genome in response to drought treatment indicate adaption to
   the local environment.^[36]9 Moreover, epigenetic variations in
   transposable element (TE) regions can be affected by environmental
   stresses.^[37]10 Silencing of the ZmMET1 gene after low-temperature
   treatment leads to demethylation of the Ac/Ds transposon region in
   maize roots.^[38]11 In sum, these results indicate an association
   between environmental stresses and DNA methylation alterations.

   DNA cytosine methylation functions as an important controller of gene
   mRNA levels. In the Arabidopsis genome, DNA cytosine methylation is
   mostly detected at CpG sites and can also occur at CHG and CHH residues
   (H indicates A, T, or C).^[39]12 In the Arabidopsis genome, most
   methylation in the gene body is detected at CpG sites, while CpG, CHH,
   and CHG site methylation occurs elsewhere and in repetitive
   regions.^[40]12 CG methylation is enriched in TEs and gene bodies, but
   non-CG methylation is mostly present in TEs.^[41]13 Often, methylation
   of gene promoter regions is believed to inhibit gene expression, while
   methylation within introns and exons can promote gene
   transcription.^[42]14 MET1 is the main CG methyltransferase, CMT3 is
   the main CHG methyltransferase, and DRM2 is the main CHH
   methyltransferase and is guided by small RNAs.

   Epigenetic features play a critical role in controlling gene expression
   and the subsequent response of an organism to its environment. As a
   major epigenetic modification, DNA methylation is not directly encoded
   in the genome sequence, and yet can modify expression and may be
   inherited for at least one generation. Several studies have shown that
   large numbers of plant genes are differentially expressed in response
   to spaceflight.^[43]4 Learning more about the spaceflight methylome of
   plants will contribute to the foundational understanding of how plants
   adapt to spaceflight. In this research, we showed the results of an
   SJ-10 spaceflight experiment. The experiment was a part of the SJ-10
   mission, an experimental project of Chinese Academy of Sciences in
   April 2016. Arabidopsis seedlings were exposed to spaceflight on board
   of SJ-10. To evaluate DNA methylome during spaceflight, we profiled DNA
   methylation genome wide in Arabidopsis seedlings. Whole-genome
   bisulfite sequencing (BS-Seq) allows DNA methylation to be measured at
   the whole-genome level with single nucleotide resolution.^[44]13 These
   results will be very useful for our understanding of the potential role
   of cytosine DNA methylation in plants adapting to the outer space
   environment.

Results

Experimental design and sample fixation on the recoverable scientific
satellite SJ-10

   The specific equipment used in the space experiment was designed and
   constructed by Shanghai Institution of Technical Physics (Fig. [45]1a,
   b). The equipment included two cultivation units, 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 the fixative unit by tube connectors. The Chinese
   recoverable scientific satellite SJ-10 was launched at 01:38 on April
   6, 2016 and landed at 16:30 on April 18, 2016. The air temperature near
   the incubator averaged approximately 23 °C. The average relative
   humidity near the incubator was approximately 25%, and the microgravity
   level was estimated to be approximately 10^−3 to 10^−4g during the
   unified flight phase.^[46]15,[47]16 Seedlings were fixed with RNAlater®
   after growth for 60 h under microgravity conditions. They were
   harvested approximately 4 h after landing, stored at 4 °C, and
   transported to our laboratory for further assays. Seedling DNA was
   extracted and used for methylome analysis. To halt cellular activities
   in space and preserve the DNA methylome profile, RNAlater® was added to
   the culture chambers on board SJ-10 and on the ground. RNAlater® has
   been experimentally verified to effectively preserve DNA and to yield
   high-quality RNA and has been used successfully in spaceflight
   applications.^[48]17 Samples of the 1 g ground controls in cultivation
   unit 1 were also fixed in RNAlater® at the same time as the in-space
   samples (Fig. [49]1d).

Fig. 1.

   [50]Fig. 1
   [51]Open in a new tab

   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 references.^[52]15,[53]16 The g-profile during launch