Patterns of Arabidopsis gene expression in the face of hypobaric stress
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Patterns of Arabidopsis gene expression in the face of hypobaric stress
Anna-Lisa Paul 2
Mingqi Zhou 2
Jordan B. Callaham 2
Matthew Reyes 1
Michael Stasiak 0
Alberto Riva 3
Agata K. Zupanska 2
Mike A. Dixon 0
Robert J. Ferl 2 3
Associate Editor: Michael B. Jackson
0 University of Guelph , Guelph, N1G 2W1 ON , Canada
1 Exploration Solutions, Inc. , Moffett Field, CA 94035 , USA
2 Program in Plant Molecular and Cellular Biology, Department of Horticultural Sciences, University of Florida , Gainesville, FL 32611 , USA
3 Interdisciplinary Centre for Biotechnology, University of Florida , Gainesville, FL 32610 , USA
Extreme hypobaria is a novel abiotic stress that is outside the evolutionary experience of terrestrial plants. In natural environments, the practical limit of atmospheric pressure experienced by higher plants is about 50 kPa or about 0.5 atmospheres; a limit that is primarily imposed by the combined stresses inherent to high altitude conditions of terrestrial mountains. However, in highly controlled chambers, and within projected extra-terrestrial greenhouses, the atmospheric pressure component can be isolated from the associated high altitude stresses such as temperature, desiccation and even hypoxia. Such chambers allow the exploration of hypobaria as a single variable that can be carried to extremes beyond what is possible in terrestrial biomes. Here, we examine the organ-specific progression of transcriptional strategies for the physiological adaptation to various degrees of hypobaric stress, as well as the response to severe hypobaria over time. An abrupt transition from a near-sea level pressure of 97 kPa to a mere 5 kPa is accompanied by the differential expression of hundreds of genes, primarily those associated with drought, hypoxia and cell wall metabolism. However, pressure transitions between these two extremes reveals that plants respond with complex, organ-specific transcriptomic responses, which also vary over time. These responses are not linear; neither with respect to the gradient of hypobaric severity from 75, 50, 25 to 10 kPa, nor with the duration of exposure of up to 3 days at 10 kPa. In the first few hours of hypobaria, plants engage changes in basic metabolism and hormonally mediated growth and development. After 12 or more hours of hypobaria, the gene expression patterns are more indicative of hypoxia and drought environmental responses. The hypobaria transcription patterns were highly organ specific, and roots appeared to be more sensitive to hypobaria than shoots in that the number of differentially expressed genes was higher in roots than in shoots. The patterns of gene expression among organs, across a gradient of atmospheric pressures and over time suggest that plants adapt to the novel stress of pure hypobaria by using recognizable metabolisms to meet appropriately interpreted hypoxia stresses, while engaging drought responses that are seemingly inappropriate to the wet and humid environment of the chambers.
Abiotic stress; Arabidopsis; hypobaria; hypoxia; Mars greenhouse; reduced atmospheric pressure
VC The Authors 2017. Published by Oxford University Press on behalf of the Annals of Botany Company.
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Current biogeographical boundaries are the result of
evolutionary selection for capacity to thrive in certain
environments. Excursion beyond those boundaries imposes
stress, which is met by further engagement of pathways
that evolved to adapt to the environment in which the
stress is grounded. We are interested in probing the
stress response contingencies that exist within plants by
using novel abiotic stresses that have not been
experienced over evolutionary time.
Hypobaria, low atmospheric pressure, is one such novel
stress. In exploration of environments beyond Earth’s
surface, altered atmospheric pressure can present both a
crucial challenge, and an operational trade space for life
. Environmental conditions in
spaceflight vehicles have traditionally been hypobaric to
reduce the masses of structural materials to load more
supplies and increase mission lengths. Concepts for
orbital and planetary greenhouses often include
reducedpressure atmospheres to decrease the costs of energy
and consumable components
(Martin and McCormick
, and the living plants within those hypobaric
habitats must be able to thrive to successfully maintain the
life support system
. Thus growing plants
in low atmospheric pressure is both a necessary
consideration for human space exploration and an opportunity for
the study of physiological adaptation contingencies.
The pressure at which plants can grow naturally on
earth is limited by physical altitude (e.g. 35
kPa—kilopascal—at the top of Mount Everest) and the temperature,
hypoxia and water stress that accompany extreme
altitudes. A combination of environmental factors makes
the natural terrestrial limit for higher plants about
50 kPa, half of the 101 kPa that defines the barometric
pressure at sea level
(Paul et al. 2004; Paul and Ferl
. Thus, an atmospheric pressure below 50 kPa
represents an environment to which no higher plant has
ever had to adapt. And yet, in artificial environments,
plants can manage quite well at pressures as low as
(Paul et al. 2004)
and there have been studies
showing that as long as the hypoxic component of
hypobaria is mitigated, seeds can germinate and then grow at
25 kPa for over a month
(He et al. 2007; He et al. 2009;
He and Davies 2012)
. This adaptability has long been the
foundation of the concept of utilizing reduced pressure
greenhouses to support human crews on the moon or
(Corey et al. 1996; Clawson et al. 1999; Fowler et al.
2000; Goto et al. 2002; Richards et al. 2006; Paul and Ferl
, and yet current understanding of the molecular
basis for plant adaptability at low pressures is limited.
In the laboratory, atmospheric pressure can be
isolated from other high altitude stresses and hypobaria
can be explored as a specific probe for examining plant
environmental response contingencies. When faced with
a novel stress, plants can respond in several conceivable
ways. They can do nothing—if there is no receptor for
the stress, nothing can be perceived to generate a
response. They can respond appropriately—if there is a
recognizable component of the stress (e.g. reduced
oxygen availability) that triggers an adaptive response. Or
they can mount an inappropriate response—if the novel
stress initiates a response that does not contribute to
survival. The latter two courses appear to manifest in the
response of Arabidopsis thaliana (Arabidopsis) to a
severe hypobaric environment. Arabidopsis responded to a
24 h hypobaric exposure (from 101 to 10 kPa), with a
unique utilization of the genome to cope with a novel
situation, a response which was not equal to a comparable
hypoxic environment at 101 kPa. 24 h at 10 kPa resulted
in the differential expression of over 200 genes in
Arabidopsis leaves, and central among these genes were
those of ABA and drought metabolisms. However, the
induction of many of the genes in this set reflects an
inappropriate response in that the plants were not actually
water stressed, yet responded as if they were by
engaging drought response metabolisms
(Paul et al. 2004)
Given the seemingly inappropriate response of 24 h
direct exposure to 10 kPa, we wished to examine the onset
and development of the response over both time and
severity of hypobaria. We also wished to examine the
organ specificity of the response to better understand the
physiological basis of the response. We present here a
compilation and comparison of the changes in gene
expression patterns that comprise the response of
Arabidopsis shoots and roots to hypobaric conditions
ranging from mild (75 kPa), moderate (50 kPa) and
severe (25 kPa) to extreme (10 and 5 kPa). We also explore
the effect of time, evaluating the gene expression
patterns as plants acclimate to extreme hypobaria (10 kPa)
after 1, 3, 6, 12, 48 and 72 h.
Reduced atmospheric pressure chambers and facilities
The Low Pressure Growth Chambers (LPGC) used in this
experiment were part of the Controlled Environment
Systems Research Facility (CESRF) at University of
Guelph, Ontario, Canada (Fig. 1A). The LPGC were
programed with variable conditions depending on each
subexperiment treatment. Each chamber was monitored at
5 min intervals and controlled for temperature, air
pressure and gas composition. Relative humidity and vapour
pressure density were also monitored. Due to the
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location of CESRF in Guelph, Ontario at 1100 feet above
sea level, the control pressure for all experimental
conditions was set at 97 kPa.
Arabidopsis thaliana (ecotype Wassilewskija) were used
throughout. Seeds were surfaced sterilized with a 5-min
rinse of 70 % Ethanol, a 50 % bleach (v/v) and Tween 20 VC
soak for 10 min, followed by multiple rinses of sterile
water. Seeds were then planted on vertically orientated Petri
plates (10 cm2) containing 0.5 MS media
, 0.45 % Phytagel (w/v) and 2.5 ppm benomyl.
Plates were sealed with Micropore (3 M) tape to allow air
(Paul et al. 2001)
Prior to atmospheric treatments, plants were grown in
an ambient pressure growth chamber at CESRF. Growth
chamber conditions were: 24-h light, 22–25 C, humidity
of 95 % or above maintained inside the Petri plates and
ambient atmosphere of 97 kPa (Guelph, ON ambient).
Experiment 1—variations on a pressure
A set of 10-day-old plants grown vertically on plates
were transferred to the LPGCs and exposed to six
different atmospheric pressures for a period of 24 h; 97, 75,
50, 25, 10 and 5 kPa. An example of how the plates were
oriented and contained within the LP chambers is shown
in Fig. 1B and C. In all treatments, the carbon dioxide
was kept consistent at a partial pressure of 0.05 kPa. In
each treatment, oxygen was kept at 21 % (v/v) of the
total chamber pressure with Nitrogen as a balance of
remaining gas. Each treatment of atmospheric pressures
and gases was replicated with three separate,
concurrently run LPGCs. Each chamber in the LPGCs contained
10 plates, with 12 plants on each plate.
Experiment 2—a 10 kPa time course
A set of 10-day-old plants grown vertically on plates
were exposed to 10 and 97 kPa in LPGCs and sampled
at six different time points. The 97 kPa atmosphere
was composed of partial pressures of 21 kPa oxygen,
0.05 kPa carbon dioxide and a balance of nitrogen. The
10 kPa samples were composed of a partial pressure of
2.1 kPa oxygen, 0.05 kPa carbon dioxide and a balance of
nitrogen. The samples were harvested at: 1, 3, 6, 12, 48
and 72 h. Each treatment of atmospheric pressures and
times were replicated with three separate, concurrently
run LPGCs. Each chamber in the LPGCs contained 10
plates of 12 plants each.
Harvest, extraction and analysis
At the end of the treatment plates were removed from
the chambers, opened and plants were harvested using
forceps directly to a fixative RNAlater (Ambion). Each
plate of 12 plants was harvested to a separate tube, for
a total of 10 tubes per treatment. To allow for later organ
specific gene expression analysis, some samples were
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separated into aerial parts (shoots) and roots before
being removed from the plate, and as quickly as possible
submerged in RNAlater to avoid wounding responses.
Samples were immediately stored according to
manufacturer recommendations, with long term storage at
20 C until extraction. A random selection of three
tubes was used from each sample group for RNA
extraction. Total RNA was extracted from the samples using
Qiashredder columns in the Qiagen RNAeasy kit and
removed residual DNA using RNase-free Dnase. RNA
concentration was determined on a BioSpectrometer
(Eppendorf) and sample quality was assessed using the
Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.).
Samples were then subjected to microarray analysis.
The 100 ng of total RNA from each sample was reverse
transcribed into double-stranded cDNA, from which
biotin-labelled cRNA was generated using the 30 IVT plus Kit
(Affymetrix). The cRNA was purified using magnetic
beads and was fragmented. Following fragmentation,
cRNA products (12.5 mg) were hybridized with rotation to
the Affymetrix GeneChip VR Arabidopsis ATH1 Genome
Arrays for 16 h at 45 C. Arrays were washed on a Fluidics
Station 450 (Affymetrix) using the Hybridization Wash
and Stain Kit (Affymetrix) and the Washing Procedure
FS450_0004. Fluorescent signals were measured with an
Affymetrix GeneChip Scanner 3000 7G. Primary data
analysis was carried out using the MAS5 algorithm within
the Affymetrix Expression Console software. Microarray
experiments were performed at the Interdisciplinary
Centre for Biotechnology Research Microarray Core,
University of Florida. Array data are available in the Gene
Expression Omnibus database with the accession
number of GSE87905.
Microarrays data analysis
The arrays (a total of 51 Affymetrix ATH1 arrays for roots
and 55 for shoots) were analysed using the R/
Bioconductor pipeline. The goal of the analysis was to
identify differentially expressed genes in each
lowpressure or low-oxygen sample versus the normal
pressure samples. The following Table 1 shows the
number of arrays in each analysed group.
The arrays were normalized using the RMA method,
and differential analysis was performed using the
‘Limma’ R package
(Ritchie et al. 2015)
. Data quality was
assessed using arrayQualityMetrics package and various
QC charts (Density & Intensity plot, NUSE, RLE and RNA
Degradation Plot). For each replicate array, each
probeset signal value from treated samples was compared to
the probe-set signal value of control samples to give
gene expression ratios. Differentially expressed genes
were identified using Limma package with a Benjamini
and Hochberg false discovery rate multiple testing
correction. Genes were considered as differentially
expressed with stringent criteria at P < 0.01, Log2 (fold
change) > 1. The P values for each treatment comparison
are included in the Supporting Information Tables S1
The differentially expressed genes in the two tissues
were clustered with the GENE-E program (http://www.
using Kendall tau distance
(Eisen et al. 1998; Saldanha
2004; Cai et al. 2012)
. Gene ontology analysis of
biological process was performed using AgriGO (http://bioinfo.
(Du et al. 2010)
terms of biological process with P < 0.01 were listed in
Supporting Information Tables S1 and S2. The pathway
enrichment analysis was performed with DAVID6.8
(https://david.ncifcrf.gov/) using Kyoto Encyclopaedia of
Genes and Genomes (KEGG) database (http://www.ge
nome.jp/sig/tool/map_pathway1.html (13 July 2017)).
Quantitative RT-PCR was used to quantify the expression
levels of genes selected from the microarrays data.
Quantitative RT-PCR reactions were conducted in
triplicate with the same RNAs as were used for the microarray
analyses, and the reactions were analysed with the
Applied Biosystems Prism 7700 Sequence Detection
(Paul et al. 2004)
. The fluorescently tagged
probes and paired primers flanking a 60–100 bp section
of the gene of interest are listed in Table 2. The gene
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Gene Name Sequence (50–30)
AHB1 (AT2G16060) AHB1-Forward GGTGGCCAAGTATGCATTGTT
expression level was normalized to a standard curve.
UBQ11 (AT4G05050) was used as the internal control.
Utilization of low-pressure environments to challenge Arabidopsis acclimation
The low pressure growth chambers (LPGC) at the
University of Guelph’s Controlled Environment Systems
Research Facility (CESRF) were configured to provide
several hypobaric atmosphere environments, including an
ambient pressure (97 kPa) control (Fig. 1A). In
Experiment 1, the plants were grown for 24 h in LPGC at
atmospheric pressures of 97, 75, 50, 25, 10 and 5 kPa. In
Experiment 2, the seedlings were grown in either a 10 or
97 kPa environment to establish a time course comprised
of 1, 3, 6, 12, 48 and 72 h. The 24 h time point was
represented in Experiment 1, and so was not repeated in the
Experiment 2 time course. These investigations were
performed in strictly monitored growth conditions;
environmental factors of light, temperature and humidity were
kept uniform among chambers. Atmospheric
composition in the chambers was kept at 0.05 kPa Carbon
Dioxide, 21 % Oxygen and a balance of Nitrogen in both
experiments regardless of total chamber pressure. As
was seen previously
(Paul et al. 2004)
, there were no
obvious morphological difference of plants grown on the
surface of MS media plates between atmospheric
conditions. All plants appeared similar in appearance to
the 97 kPa control and even plants grown in extreme
hypobaria (10 and 5 kPa) showed no physical signs of
desiccation, such as wilting. Figure 2 provides
representative images of plants after each treatment, just prior to
being harvested to RNAlater. Figure 2A shows a
representative plate of 10-day-old plants after 24 h of
exposure to 97, 75, 50, 25, 10 and 5 kPa. Figure 2B shows a
representative plate of 10 day old plants after being
grown in either a 10 or a 97 kPa environment for 1, 3, 6,
12, 48 and 72 h. One additional atmospheric treatment
was made at 0.7 kPa, a pressure that is comparable to
what is typical on the surface of Mars. The plates of
plants were observed in real time and then were allowed
to recover and planted in soil. Figure 2C shows a picture
of the plate of wilted plants after 24 h at 0.7 kPa (left),
alongside a photograph of the transplanted seedlings
2 weeks after being transferred to soil (right). The plants
recovered from the severe wilting imposed by 0.7 kPa
and went on to flower and eventually set seed.
Overview of global transcriptional responses to different levels of hypobaria—experiment 1
The hypobaric transcriptomes of Arabidopsis roots and
shoots were evaluated in response to 24 h of treatments
in mild (75 kPa), moderate (50 kPa), severe (25 kPa) and
extreme (10 and 5 kPa) low atmospheric pressures
(Fig. 3). The genome-wide relative expression patterns
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from plants in each hypobaric condition were compared
to the reference transcriptome from plants grown in a
chamber kept at ambient pressure (97 kPa). Unless noted
otherwise, in all cases gene expression showing at least
2-fold change in abundance and P < 0.01 were defined
as differentially expressed genes. A total of 3156 genes
were differentially expressed by at least 2-fold in at least
one hypobaric treatment, and yet not a single gene was
co-ordinately expressed among all of the treatments in
leaves, and only three genes showed coordinate
expression in roots: POX1 (Proline dehydrogenase 1), PI4KG5
(Phosphatidylinositol 4-kinase gamma 5) and a gene
(AT1G78850) that encodes a curculin-like lectin [see
Supporting Information—Table S1].
For treatments at pressures between 75 and 10 kPa,
the transcriptomes showed dramatic tissue-specific
patterns; roots shared few differentially expressed genes in
common with shoots in response to hypobaria.
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In addition, roots appeared more sensitive to these
environmental changes, as far more genes were significantly
changed in roots than in shoots (Figs 3 and 4A) [see
Supporting Information—Table S1]. In the very extreme
hypobaria of 5 kPa, there was more overlap in gene
expression patterns between roots and shoots, with many
more genes being differentially expressed in this
environment than any other hypobaric treatment (Fig. 4A) [see
Supporting Information—Table S1]. Supporting
Information Table S1 provides a list of differentially
expressed genes and GO terms of biological process in
response to each hypobaric condition.
For roots, the differential expression patterns in 75 kPa
were quite distinct when compared with other
conditions. This mild hypobaria activated genes associated
with transportation and localization processes, whereas
moderate and severe hypobaria (50 kPa through 5 kPa)
gene expression patterns were dominated by genes
typically associated with abiotic, osmotic and oxidative
stress responses, and the abundance of differentially
expressed genes in these functional categories increased
with the severity of the hypobaric environment. This
trend is particularly evident in genes associated with
hypoxia, such as SUS1, SUS4, PDC1, PDC2, AHB1, ERF073,
GLB3, LDH, PCO1 and ADH1
(e.g. Bailey-Serres and Chang
2005; Chang and Meyerowitz 1986; Gasch et al. 2016;
Gibbs et al. 2011; Mustroph et al. 2010; Vartapetian and
and with defence responses, such as
represented by the XTH gene family (e.g. Eklof and Brumer
Le Gall et al. 2015
Most of the genes that are repressed in hypobaric
environments are associated with growth and metabolism.
The mild hypobaria of 75 kPa resulted in more
downregulated genes than 50 and 25 kPa, among which there
was only a small overlap (Fig. 4B) [see Supporting
In shoots, many of the differentially expressed genes
at 50 kPa are associated with transport functions (e.g.
LTH7, CAX3, STP13 and SWEET10) and pathogen
response (e.g. THI2, WRKY18 and TAT3) rather than
hypoxia. Below 50 kPa, the induction of genes associated
with desiccation and water deprivation responsive
pathways predominated. The Dehydration-Responsive
Element-Binding Factor 2A (DREB2A)
(Sakuma et al.
as well as marker target genes RD29A, KIN1,
COR47, COR15a, COR15b and ERD10
(Kasuga et al. 1999;
Seki et al. 2002; Kimura et al. 2003)
were induced by
hypobaric environments of 10 kPa and below in shoots,
but not by less severe hypobaria (Fig. 3). In addition,
several members of PYL ABA receptor family were also
involved in the effects of extreme hypobaria (Fig. 3) [see
Supporting Information—Table S1]. For instance, PYL5
and PYL8 expression levels were significantly enhanced
in roots in both 10 and 5 kPa and repressed in shoots in
5 kPa. An examination of genes that are induced by ABA
in guard cells
(Leonhardt et al. 2004)
reveals many of
which were also induced in shoots in 5 kPa (PP2CA,
ATHB-12 and XERO2). Compared with 10 kPa, 5kPa
elicited huge changes including hundreds of genes that
were not significantly affected in other pressures (Fig. 3)
[see Supporting Information—Table S1].
Time course-dependent transcriptome analysis revealed an organ specific pattern in response to extreme hypobaric stress—experiment 2
The atmospheric environment of 10 kPa was chosen to
evaluate the progression of the physiological adaptation
to hypobaria. Transcriptome analyses of 10-day-old
plants (at start of the experiment) exposed for 1, 3, 6, 12,
48 and 72 h revealed organ-specific and time point
dependent patterns of gene expression (Fig. 5). Since the
10 kPa, 24 h treatment was represented in Experiment 1,
this treatment was not included in Experiment 2. There
were a total of 2801 genes that were differentially
expressed in at least one time point over the course of the
3 day experiment. Only 200 of those genes (7 %) were
coordinately expressed with similar trends in both roots
and shoots [see Supporting Information—Table S2].
The consistently up-regulated genes included typical
hypoxia responsive genes such as ADH1, AHB1, PDC1,
PDC2, PCO1 and PCO2
(Chang and Meyerowitz 1986;
Gibbs et al. 2011; Gasch et al. 2016)
as wells as drought
and cold marker genes like RD29A and KIN1
(Yamaguchi-Shinozaki and Shinozaki 1994; Wang and
Cutler 1995; Wang et al. 1995; Narusaka et al. 2003)
In clustering analysis, Kendall tau distances of gene
expression between each time point were used to show
the similarity of transcriptome responses in time course.
In roots, the patterns were distinctly different in each of
the early time points (1 and 3 h), but then a trend
developed at 6 h that was continued through 72 h. In shoots,
it was the earlier time points that exhibited similar
patterns of gene expression (from 1 to 12 h), while in the
later time points new sets of genes were either induced
or repressed (Fig. 6A). The relative intensity of the
response was gauged by the number of differentially
expressed genes, and their relative increase or decrease at
each time point. In roots, there appears to be a peak of
differential expression at 12 h, with a relatively smooth
increase approaching 12 h and then a decline
approaching the 72 h point (Fig. 6B). In shoots, there is not a clear
trend, other than the overall number of differentially
expressed genes tends to increase over time (Fig. 6C).
Roots respond more quickly than shoots, as
demonstrated by the large number of induced genes at 1 h. In
contrast, the intensity of the response appears to
generally mount over time in shoots, particularly with respect
to down-regulated genes. The early response in roots
could be promoted by the hypoxic component of
hypobaria; the plant low-oxygen response consists of
approximately two stages in a previous time course study,
0–0.5 h and 2–20 h
(Klok et al. 2002)
. Indeed, some key
hypoxia responsive genes were induced and kept active
while other stress responders like WRKY6
(Skibbe et al.
2008; Chen et al. 2009)
and WRKY21 (Phelps-Durr et al.
2005) were transiently induced at 1 h. At time point of
3 h, cell wall organization and cell growth associated
genes such as gibberellin biosynthesis gene GA3ox1
(Arnaud et al. 2010)
were transiently repressed.
Metabolic transportation and localization related genes
were induced at 6 h and kept active through the 72 h
time point. From 12 to 72 h the cellular metabolic
processes appeared to be continually altered, as for each
time point a unique set of genes were differently
expressed within this functional category (Figs 5 and 6B).
Shoots exhibited fewer differentially expressed genes
in the early time points than roots. Compared to roots,
shoots also have fewer examples of genes which display
a mounting degree of expression over time. Nonetheless,
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there were many genes associated with biosynthetic and
metabolic process that were induced or repressed early,
and maintained or exceeded those levels of expression
over time (Figs 5 and 6C). At 48 h, additional water stress
and ABA responsive genes such as PP2C family members
HAI1, ABI2 and homeodomain leucine-zipper gene
(Soderman et al. 1996; Merlot et al. 2001; Olsson
et al. 2004; Yoshida et al. 2006)
were involved to cope
with hypobaria. Finally at 72 h, stress response was
down-regulated and nitrogen compound metabolic
process was induced [see Supporting Information—
In pathway enrichment investigation, the scheme of
metabolic changes in roots and shoots could be further
exhibited (Fig. 7). Different from the GO analysis using
time course-dependent group of genes shown in
Supporting Information—Table S2, Fig. 7 shows the
KEGG pathways significantly enriched in all differentially
expressed genes (Benjamini corrected p-value < 0.05) of
each time point. Metabolic changes widely happened in
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both two tissues. Roots showed more similar metabolites
biosynthesis process before 12 h, and from 12 to 72 h
nitrogen and hormone associated signalling were
identified. In shoots, no pathway was detected in 1 h, and
multiple secondary metabolites associated signalling
changed after 12 h (Fig. 7).
Validation of array data using qRT-PCR
In this study, we used unadjusted P value to perform
differential analysis, for the adjustment such as FDR would
hide all differentially expressed genes in some
comparisons. For validation of array data, two hypoxia
associated genes AHB1 (AT2G16060) and PDC1 (AT4G33070)
as well as two drought associated genes RD29A
(AT5G52310) and RD20 (AT2G33380) were subjected to
qRT-PCR analysis. Most of the expression changes
showed an agreement between qRT-PCR and RNA-seq
data (Fig. 8). AHB1 and PDC1 with P values < 0.01 but
with FDR > 0.05 in the arrays exhibited significant
changes in 50 kPa in roots, indicating that FDR indeed
limited the identification of differentially expressed
genes in our data. Similarly, the desiccation responsive
genes RD29A and RD20 were highly up-regulated in
shoots in responses to extreme hypobaric stress.
It is known that plants survive and thrive in hypobaric
environments according to the extensive studies that have
focused on direct physiological changes and metabolic
impact of low atmospheric pressures on plants
Staby 1981; Musgrave et al. 1988; Andre and Massimino
1992; Daunicht and Brinkjans 1996; Corey et al. 2002;
Spanarkel and Drew 2002; He et al. 2003)
presented here suggest, however, that plants mount an
extensive and complex stress response at the level of gene
expression in order to drive that survival—a response
that is not linear with respect to increasing severity, and
has dramatic organ specificity. This stress response
profile seems contradictory to the apparent ability of plants
to thrive at low pressures, and allows an examination of
the value and appropriateness of the responses to the
novel stress of hypobaria.
Two highly recognizable stressors of hypobaric
environments are the reduction of available oxygen and
elevation of evapotranspiration rates
(Paul et al. 2004;
Rygalov et al. 2004)
. Genes associated with both hypoxic
stress and drought responses are highly represented in
even mild hypobaria. However, while mitigating the
hypoxia characteristic of hypobaric environments makes
metabolic sense, engaging extensive drought responses
is inconsistent with the well-watered and humid
environment in these experiments. Therefore, the drought
responses may be a metabolically inappropriate reaction
and therefore present an unnecessary metabolic cost in
low pressure environments, especially the mild and
moderate pressures of 25 kPa and above.
In terms of atmospheric pressure, the habitable
terrestrial limit for angiosperms converges on about 50 kPa
(Ko¨rner 2003a; Klimes 2003; Namgail et al. 2012)
, a value
that is driven by the limits of habitable altitudes in
mountains of neo-tropical latitudes, such as the Himalayas.
The physical terrestrial limit (as in ground to stand on) of
atmospheric pressure is 34 kPa, which can be found at
the top of Mt. Everest. In natural habitats, limits are
primarily driven by combinations of extreme temperature
and water stress, rather than by atmospheric pressure.
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However, in laboratory chambers the harsh
environmental characteristics that attend high altitudes can be
mitigated (75 and 50 kPa), and truly novel pressure
environments (25, 10, and 5 kPa) can be created that are
survivable by terrestrial plants. These capabilities
allowed us to explore the fundamental limits of plant
responses to novel environments, but also to examine the
mechanisms that underlie coping with only the
hypobaric component of high altitude environments.
The degree of hypobaria greatly influences the
transcriptional response of plants, but the response is not
linear. Of the more than 3000 genes that are differentially
expressed among the five hypobaric treatments (Fig. 2)
[see Supporting Information—Table S1], only three in
roots (POX1, PI4KG5 and an unknown D-mannose
binding lectin protein) and two in shoots (PCR1 and GSTF6)
are coordinately induced across all hypobaria conditions.
These genes may play basic roles in plant hypobaric
responses although little is known about their molecular
functions. The primary reason behind the virtual absence
of coordinately expressed genes among treatments is
that the gene expression profile of the 75 kPa treatment
had virtually nothing in common with hypobaric
treatments of 50 kPa and lower. If the 75 kPa data are
considered separately from the other treatments, then a
pattern begins to emerge from the transcriptional
response to increasingly severe hypobaric exposure.
Increasing the severity of hypobaric environments
Although 75 kPa would seem to be simply a more benign
environment than the other atmospheres, there are
characteristics of 75 kPa that set this pressure apart from
the rest. Of the five treatments, 75 kPa is the only
pressure that represents a hypobaric environment that can
be part of a biologically rich terrestrial habitat. In other
words, 75 kPa does not present a novel environment that
is outside the evolutionary experience of higher plants; it
can be found on the slopes of a 3000 m mountain. In
addition, the combination of reduced O2 and CO2 partial
pressures, reduced boundary layer resistance and
increased gas diffusion rates, can increase photosynthetic
efficiency in plants grown at 75 kPa
(He et al. 2013;
Richards et al. 2006; Rygalov et al. 2004)
, and change the
efficacy hormones such as ethylene
(Apelbaum and Burg
1972; Burg and Burg 1965)
. Further, although effects
vary widely among plant species and habitats, there is
ample evidence that photosynthetic efficiency is
influenced by altitude in natural hypobaric environments
(Gale 1972, 1973, 2004; Shi et al. 2006)
. Thus, the
evolutionarily familiar features of the 75 kPa environments
engage metabolic processes that are familiar and
reasonably well understood.
In the mild hypobaria of 75 kPa, a common theme
among the most highly induced genes in roots was the
VC The Authors 2017
over expression of genes encoding transcription factors
and genes associated with root development or cell
growth. Most of the highly down regulated genes in roots
are associated with light signalling and the
photosystems, while very few are typically associated with stress
metabolism. In shoots, there was an abundance of
differentially expressed genes associated with oxidative
stress and pathogen responses, as well as many genes
encoding transporters. Many of the down regulated
genes are associated with cell growth and elongation.
In contrast to the familiar and well understood
responses at 75 kPa, responses to 50 kPa are distinctly
unfamiliar and seemingly illogical. The transition to 50 kPa
marks the limits of terrestrial plant environments, yet
although this is a harsher hypobaric treatment, plants
respond with a decrease in the number of differentially
expressed genes (112 in roots and 23 in shoots)
compared to the 75 kPa response (196 in roots and 60 in
shoots). More interestingly, the type of genes that are
expressed at 50 kPa are quite different than those at 75 kPa
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and they represent a developing pattern of response
that is carried though with increasing severity of
hypobaria, particularly in roots. Figure 3 provides a sense of this
transition pattern. About a third (35) of the differentially
expressed genes at 50 kPa in roots are coordinately
expressed in 25, 10 and 5 kPa. Most are upregulated, and
many are indicative of hypoxic stress.
Below 50 kPa, decreasing the atmospheric pressure
correlates directly with an increase in the number of
differentially expressed genes in an organ-specific manner.
In roots there is substantial (60–70 %) overlap of the
genes that are induced in each step of severity, giving
the impression that the root response to increasingly
severe hypobaria builds on a common metabolic
foundation (Fig. 2) [see Supporting Information—Table S1]. In
roots, it appears that a consistent core of the response to
increasingly reduced atmospheric pressure is an
induction of genes associated with hypoxic stress.
In shoots, the patterns of gene differential gene
expression are more varied among hypobaric treatments,
and hypoxic stress associated genes are less abundant
than in roots at the same pressure. One of the mitigating
effects of hypoxic stress in shoots is the presence of
(e.g. Juntawong and Bailey-Serres 2012)
largest category of differentially expressed genes in
shoots from 10 and 5 kPa are those associated with
desiccation and ABA signalling related processes. For
instance, in the top 25 most highly induced genes in
10 kPa, almost a third fall into this category (e.g. LTP3,
LTP4, LEA7, BG2, PXG3, COR15A, COR15B and STR17).
But are hypobaric plants ‘drought stressed’ from a
water deficiency? The plants were grown in a humid
environment (>95 % rh within the plates), on a medium that
provided a continuous supply of water, and although
fresh weights were not collected from treatment and
control representatives in these plants, the plants did not
show any outward signs of dehydration stress, such as
wilting. This observational conclusion is supported by
fresh weight data collected in an earlier experiment, in
which there was no significant change in average fresh
weight between hypobaric and control Petri plate-grown
plants after 24 h at 10 kPa
(Paul et al. 2004)
. Thus it is
likely that the desiccation response is due to the
perception of increased water flux caused by the low pressure
environment rather than the absolute loss of water in
(Corey et al. 2002; Paul et al. 2004)
Correspondingly, the drought response is related to the
rate of water movement through the leaves, as a few of
key desiccation associated genes including RD29a,
COR15a and KIN1 (Kasuga et al. 1999) were only induced
in response to extreme hypobaric stress (below 25 kPa).
The sense of water stress by plants in mild, moderate
and even severe low atmospheric pressure around
25 kPa can be quite weak. In addition, there is a marked
difference in the induction of drought-induced genes in
roots as compared with shoots. Although some of the
hallmark genes (e.g. COR78 and COR15A
(Horvath et al.
1993; Wilhelm and Thomashow 1993)
are also induced
(Zhou et al. 2011)
, the profiles in leaves and roots
are more closely aligned in response to drought stress.
Increasing the duration in a severe of hypobaric environment
The 10 kPa hypobaric environment was selected to
explore the molecular processes associated with the
physiological adaptation of Arabidopsis to extreme hypobaria
over time. Plants grown at 10 kPa for 24 h present a strong
transcriptional response, and distinct organ-specific
patterns of gene expression in response to hypobaria (Fig. 5).
The time course was constructed to characterize early
responses (1, 3, 6 and 12h of exposure) and then longer
(48 and 72 h) to discover responses that may represent
an approach to homeostasis.
Using clustering and pathway enrichment analysis, we
observed that roots and shoots underwent different
stages in the duration of hypobaric treatment. These
gene expressions provided a preliminary look into how
plants physiologically adapt to the novel environment of
10 kPa. Initially, both roots and shoots appeared to
adjust basic metabolic processes; enhancing carbohydrate
metabolism while aspects of growth and development.
It appeared to take about 72 h in 10 kPa for plants to
have stabilized many of the initially adjusted metabolic
processes, as many of the early onset gene inductions
and repressions were no longer differentially expressed.
After 72 h, there was an increase in differential
expression of genes associated with surviving aspects of
drought and other abiotic stress, particularly hypoxia,
which was consistent with the impact of the hypoxic
component of hypobaria. However, there were distinct
differences in how roots and shoots each responded to
hypobaria over time [see Supporting Information—
In roots, although the overall expression patterns of 1
and 3 h were comparatively distinct and the transcription
time points after 6 h were more closely aligned (Fig. 6A),
the 12 h point in the time course appeared to mark a
boundary for a different set of metabolic pathways (Fig.
7). The initial response in roots appeared to primarily
adjust in numerous metabolic processes such as glycolysis
changes, which is a typical hypoxia related metabolic
(Liu et al. 2005)
. After 12 h, gene expression
patterns suggested an increase in plant hormone signalling
changes while many processes associated with growth
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and development were negatively regulated [see
Supporting Information—Table S2].
In shoots, the gene expression patterns from 1 to 12 h
were more similar while the late stage of 48 and 72 h
showed distinct patterns (Figs 5 and 6A). Accordingly,
the metabolic pathway identification was in line with the
12 h time point of boundary (Fig. 7). From the onset of
hypobaria to 12 h, gene expression patterns suggested
that shoots were engaging multiple stress responses
while increasing cellular metabolism such as
alphaLinolenic acid metabolism. At later stages, the
abundance of differentially expressed genes associated with
desiccation and drought signal transduction further
suggested that plants were attempting to physiologically
adapt to perceived desiccation. In summary, the time
point of 12 h can be a turning point of plant metabolism
in responses to extremely low atmospheric pressure.
Although the transcriptomic response of roots and
shoot to each hypobaric time point were mostly distinct
with respect to specific genes, there were several cases
where different members of the same gene family were
engaged in an organ-specific manner. In other words,
although the exact same genes were not coordinately
expressed across the board in both roots and shoots,
representatives of the same gene family were engaged
in both organs. Two interesting examples are the PLY
family of ABA receptors, and the XTH family of cell wall
The PYL family of ABA receptors serve to play a major
role in ABA signalling required for vegetative and
reproductive growth such as quantitative regulation of
stomatal aperture in leaves, and yet the 14 members of the
family can have distinct functional differences
(Gonzalez-Guzman et al. 2012)
. In roots, receptor
components PYL2, PYL5, PYL6 and PYL8 were up-regulated
especially in early stage (3 and 6 h), while PYL1 was
down-regulated along every point of time course.
However in shoots, the genes encoding PYL5 and PYL7
were down-regulated in early stage (before 6 h). The
down-regulation of PYL5 and PYL7 in shoots seems to
run counter to a hypobaric adaptive response as it
should render the plants more sensitive to water loss by
increasing the stomata aperture, and thereby enhance
the rate of hypobaria-induced water movement from the
plant. However, it is possible that roots are inducing
PYL’s as part of a plant-wide ‘drought’ response
interpreted from the elevated water movement through the
stomata, but that additional sensing mechanisms (such
as turgor receptors) provide feedback in leaves that
influences gene expression in the leaves
The XTH family of enzymes function in xyloglucan
endotransglucosylation (XET) and xyloglucan hydrolysis
(XEH) to regulate chain length and modifications of
xyloglucans. Xyloglucans are hemicellulose structural
molecules that coat and cross-link cellulose fibers in the
primary cell wall and are particularly important in
maintaining the structural integrity to withstand high turgor
pressure within cells conducting water (such as phloem
(Bourquin et al. 2002)
and have a wide a
varied distribution among plant organs
. Nineteen members of the XTH family
were differentially expressed in response to hypobaria in
both roots and shoots: XTH5, XTH7, XTH8, XTH12, XTH13,
XTH14, XTH15, XTH16, XTH17, XTH18, XTH19, XTH20,
XTH21, XTH22, XTH23, XTH24, XTH25, XTH30 and XTH31,
but in widely differing patterns. In roots, XTH7, XTH12,
XTH13, XTH14, XTH16, XTH17 and XTH21 were generally
downregulated, and XTH5, XTH18, XTH20, XTH22 and
XTH23 were generally upregulated. A few were varied in
their expression patterns across the time course (XTH15
and XTH24). In shoots, most of the differentially
expressed XTH’s were down regulated after 6 h at 10 kPa
(XTH4, XTH8, XTH15, XTH19, XTH25 and XTH30). The
exception was XTH31, which was upregulated after 3 h, in
contrast to what was seen in roots for XTH31.
The differences between root and shoot utilization
implied that each organ was using diverse cell wall
metabolic tools, respectively, to sense and respond to the
extreme hypobaria. The enzymes that regulate features
of these structural molecules are of particular interest
because of the flexibility they can confer in physiological
adaptation to stress by providing a mechanism to
regulate cell wall expansion and structural integrity in
response to environmental changes
(Rose et al. 2002; Eklof
and Brumer 2010)
. It is possible that this family of cell
wall remodelling genes plays a substantial role in the
rapid adjustment of cell wall structure to accommodate
changes in the environment, particularly those
associated with water stress.
Interestingly, examples from the extended family of
cell wall remodelling genes are also abundant in the
differentially expressed genes in response to another novel
environment—spaceflight. There have been several
recent spaceflight transcriptome studies that have shown
that genes associated with cell wall remodelling are
important to the physiological adaptation to this novel
environment as well
(Paul et al. 2012, 2013; Correll et al.
2013; Sugimoto et al. 2014, Kwon et al. 2015)
. It is
possible that the cell wall, as the first line of defence for a
plant cell, is also the first responder to environments
that present a novel stimulus. Both spaceflight and
hypobaria may engage sensing mechanisms that can be
interpreted as effecting a breach or a loosening of cell wall
structures, which in turn engage a variety of genes that
typically target pathogen responses and mechanical
wounding. While both hypobaric and spaceflight
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responses are highly organ specific in nature (Figs 3 and
(Paul et al. 2013; Zhou et al. 2017)
, organ specific
responses are often a characteristic of plant stress
(e.g. Mustroph et al. 2014; Narsai et al 2010)
These studies all suggest that the strategies used to
adjust to a new or challenging environment tailored to the
metabolic background of a given plant organ or cell type.
The unique differential gene expression patterns in
response to hypobaria suggest that the physiological
adaptation of Arabidopsis to reduced atmospheric pressure
is more complex than the acclimation to the reduced
partial pressures of oxygen inherent to low atmospheric
pressures. Arabidopsis plants respond to hypobaria with
a variety of changes in gene expression patterns that are
organ specific, change with respect to the severity of the
hypobaric environment, and adjust over time. The
hypobaric response appears to overlay the perception of
desiccation and biotic challenge on top of the perceptions of
hypoxic stress, which suggests that the practical
compensation for hypobaric stress in plants cannot be as
simple as increasing the oxygen content, as is done for
humans in the low pressure environments of space
vehicles and space suits, or on the top of Mt. Everest
and Ferl 2006)
These studies provide a window into the nature of the
strategies used for physiological adaptation to hypobaria
as an example of novel environments, and can allow
dissection of specific components of plant responses to
hypobaria as part of space exploration mission habitat
design. For example, in a well hydrated, yet hypobaric
greenhouse on Mars, plants would not need to expend
the metabolic energy to maintain the drought response
the hypobaria would elicit. Thus, there could be an
advantage to engineer the plants along with the habitats
envisioned for planetary exploration. The more
fundamental application of this physical phenomenon is that
plant metabolic responses to drought stress and
stomatal regulation can be examined without subjecting the
plant to comprehensive desiccation.
In addition, these transcriptome analyses may reveal
genes that have contributed to the natural adaptation of
plants to high altitude environments. It has been argued
that plants that have evolved to live in a ‘stressful’
environment are not actually ‘stressed’ at all, rather, this is
the habitat that is normal to them, and so the metabolic
processes employed are just as benign as those engaged
by their cousins living in seemingly more hospitable
(e.g. Ko¨rner 2003b)
. Indeed, many of the
differentially expressed genes at 75 kPa (e.g. genes encoding
gibberellin-regulated proteins, xyloglucan
endotransglucosylase/hydrolases, glutathione s-transferases, dehydr
ins and expansins) are also differentially regulated in high
altitude species and ecotypes when compared to
representatives indigenous to lower altitudes
et al. 2013; Dogra et al. 2016; Luo et al. 2015)
As a unique stress, hypobaria presents an opportunity
to understand the development of plant responses to
novel environmental stress. We conclude that some
aspects of the overall response to hypobaria are
appropriate, in that hypoxia responses to the low oxygen of
hypobaria make sense. However, we also conclude that
some elements of the response are inappropriate, in that
the drought responses that are seen in hypobaria are
inconsistent with a wet and humid environment. These
seemingly inappropriate responses may help illuminate
drought perception mechanism in plants, and further
suggest strategies for improving growth in hypobaria
especially as pressure is considered in extraterrestrial
habitats and vehicles.
The following additional information is available in the
online version of this article—
Table S1. Full list of differentially expressed genes in
response to 75 kPa, 50 kPa, 25 kPa, 10 kPa and 5 kPa in
roots and shoots of 10-day-old plants. There are 3156
genes that present significant (p < 0.01) differential
expression by at least 2-fold in at least one condition
(including 75 kPa vs 97 kPa, 50 kPa vs 97 kPa, 25 kPa vs
97 kPa, 10 kPa vs 97 kPa and 5 kPa vs 97 kPa in roots or
shoots). The differentially expressed genes are
categorized according to log value of fold change and GO terms
of biological process are listed for each gene clade.
Table S2. Full list of differentially expressed genes in
response to 10 kPa during a time course in roots and
shoots of 10-day-old plants. There are 2801 genes that
present significant (p < 0.01) differential expression by at
least 2-fold in at least one condition (including 10 kPa for
1 h vs 97 kPa for 1 h, 10 kPa for 3 h vs 97 kPa for 3 h,
10 kPa for 6 h vs 97 kPa for 6 h, 10 kPa for 12 h vs 97 kPa
for 12 h, 10 kPa for 48 h vs 97 kPa for 48 h and 10 kPa for
72 h vs 97 kPa for 72 h in roots or shoots). The
differentially expressed genes are categorized according to log
value of fold change and GO terms of biological process
are listed for each gene clade.
Sources of Funding
This work was funded by NASA grants NNX13AM46G and
NNA04CC61G to R.J. Ferl and A.-L. Paul.
VC The Authors 2017
Contributions by the Authors
A.-L.P. and M.Z. contributed equally to the manuscript.
A.-L.P. and M.Z. performed the data analysis, and took
the lead on manuscript development. A.-L.P, R.J.F., J.B.C.,
M.R. and M.S. prepared plant materials and conducted
the low atmospheric pressure treatments at the
University of Guelph. A.R. carried out statistical analysis
of array data. A.K.Z. contributed to data analyses and
was responsible data archiving in GEO. M.A.D. is the
director of the University of Guelph Controlled
Environment Systems Research Facility, and contributed
to the experimental design. R.J.F. and A.-L.P. were
responsible for the overall experimental design and
conduct of the experiments. All authors read and approved
the final manuscript.
Conflict of Interest Statement
The authors thank the members of the UF SpacePlants
Lab, the UF ICBR Bioinformatics Core and the University
of Guelph Controlled Environment Systems Research
Facility, for their support. This work was funded by grants
NNX13AM46G and NNA04CC61G to R.J. Ferl and A-L. Paul
from NASA Space Life and Physical Sciences managed
through Kennedy Space Centre.
VC The Authors 2017
VC The Authors 2017
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