the biology of low atmospheric pressure – implications...

Gravitational and Space Biology 19(2) August 2006 3

THE BIOLOGY OF LOW ATMOSPHERIC PRESSURE – IMPLICATIONS FOR EXPLORATION

MISSION DESIGN AND ADVANCED LIFE SUPPORT

Anna-Lisa Paul and Robert J. Ferl

University of Florida, Gainesville, FL

ABSTRACT

Atmospheric pressure is a variable that has been often

manipulated in the trade space surrounding the design and

engineering of space exploration vehicles and extraterrestrial

habitats. Low pressures were used to reduce structural

engineering and launch mass throughout the early human space

program; moreover, low pressures will certainly be considered

in future concepts for the same reasons. Fundamental

understanding of the biological impact of low pressure

environments is therefore critical for the successful

consideration of this variable, being particularly important when

considering future, potentially complex bioregenerative life

support systems. However, low pressure biological effects are

also critical considerations that should be incorporated into near

term vehicle designs, designs that may set hardware and

operations criteria that would carry over into far-term future

designs.

In order to begin to define the fundamental biological responses

to low atmospheric pressure, we have identified the molecular

genetic responses central to the initial exposure of the model

plant Arabidopsis to hypobaric stress. Less than half of the

genes induced by hypobaria are induced by hypoxia,

establishing that response to hypobaria is a unique biological

response and is more complex than just an adaptation to low

partial pressures of oxygen. In addition, the suites of genes

induced by hypobaria confirm that water movement is a

paramount issue in plants. Current experiments examine gene

expression profiles in response to a wide variety of pressures,

ranging from slight to extreme hypobaria. Results indicate that

even small changes in atmospheric pressure have attendant

biological consequences deserving consideration during the

concept and design of vehicles and habitats. Moreover, the range

of pressures to which plants can adapt suggests that very low

pressures can be considered for plant-specific habitats.

The choices of atmospheric pressure within spaceflight and

extraterrestrial habitats are not merely engineering

considerations but are biological considerations of the highest

order, and modern molecular tools can be employed to increase

understanding of the biological consequences of pressure

engineering decisions.

INTRODUCTION

An almost bewildering myriad of environmental

parameters have been presented to Terran life forms

during the process of evolutionary change. Life has

therefore adapted and colonized a vast variety of

environments and many of those environments contain

extremes of one parameter or another; however, extreme

terrestrial altitude has escaped colonization by higher life

forms because of a suite of parameters that together

prevent habitability. Atmospheric pressure is one of the

major parameters that limit life to lower earth altitudes.

Figure 1. The relationship between altitude and atmospheric

pressure. As the elevation increases from sea level the

atmospheric pressure decreases. The range of the earth’s

atmosphere is 101 kPa at sea level to near 0 kPa at 30,000 m.

The current atmospheric attributes of the Earth’s surface

are the culmination of the physics of our planet and the

impact of over a billion years of biology and geology.

Biology has evolved and expanded into a wide range of

environments, including those that press the limits of

terrestrial altitudes, where physiology is limited by the

extremes of temperature, moisture and atmospheric

pressure that are the intrinsic components of terrestrial

high altitudes (Figure 1). Indeed, it is only where all three

of these extremes converge that we see an absence of life

on our planet. On tropical mountains mammals and large

plants (e.g. hyraxes and giant lobelias of Kilimanjaro) are

found no higher than altitudes of 5000 m (Njiro, 2005)

and herbaceous plants no higher than 5600 m (Körner,

2003). Yet even these altitude limits are dependent upon

other environmental variables. For example, the upper

limit of forests in tropical mountains may be 4000 m, yet

that same limit can be less than 2000 m in more temperate

latitudes (Körner and Paulsen, 2004). In addition, for

mammals and birds, the limits of typical habitation appear

to be delimitated as much by easy access to food and

available oxygen as by temperature, moisture and

atmospheric pressure. For humans, this habitation limit is

around 4200 m in the latitude of the Himalayas, where the

village of Kibber, India, is located (Table 1). In general,

human excursions to higher altitudes and lower pressures

____________________

* Correspondence to: Robert J. Ferl Horticultural Sciences Department

University of Florida

Gainesville, FL 32611-0690

Email: [email protected]

Phone: 352-192-1928x301; Fax: 352-392-4072

A-L. Paul and R. J. Ferl — Biology in Low Atmospheric Pressure

4 Gravitational and Space Biology 19(2) August 2006

requires supplemental oxygen – though oxygen alone

cannot alleviate all difficulties for humans at low

pressures and high altitudes (Maggiorini et al., 2001;

Bartsch et al., 2005).

In natural environments, plant growth at high altitudes is

more limited by temperature than pressure; the 5600 m

limit on Kilimanjaro is not imposed by the atmospheric

pressure but rather by the fact the ground freezes every

night. Laboratory experiments indicate that if plants are

kept from freezing and are provided with adequate water,

they can be maintained at pressures far less than that

present at the summit of Kilimanjaro (e.g. Mansell et al.,

1968; Gale, 1973; Boston, 1981; Rule and Staby, 1981;

Andre and Richaux, 1986; Musgrave et al., 1988a; Andre

and Massimino, 1992; Daunicht and Brinkjans, 1992;

Ohta et al., 1993; Corey et al., 1996; Iwabuchi et al.,

1996; Corey et al., 2000; Ferl et al., 2002; Goto et al.,

2002; He et al., 2003; Paul et al., 2004). It is only in

artificial environments, such as those of the human

spaceflight program, that atmospheric pressure becomes a

variable independent of the temperature, moisture and gas

composition concerns that accompany terrestrial altitudes.

In the contained, closed, and engineered volumes of

extraterrestrial habitats and vehicles, challenges are

created by the need to contain atmospheric pressure

against the vacuum of space. Hence, atmospheric pressure

has been independently manipulated to levels well beyond

the limits imposed by altitude conditions on Earth.

The idea that plants can be successfully cultured in very

low atmospheric pressures for the purposes of advanced

life support in non-terrestrial environments has serious

implications for attaining the goal of taking humans to

new planetary surfaces. A primary and long-term goal of

sustaining life in remote space locations is to minimize

the amount of mass, and therefore energy, required to

launch and maintain life support systems. Furthermore, if

one considers maximizing the use of local resources, then

it would be desirable to make use of ambient light, which

makes it necessary to have a structure with maximum

transparency. This then leads to the question of what

materials would be both sufficiently transparent and

sufficiently strong to contain a plant growth atmosphere

that would sustain a higher pressure than the near vacuum

present on the Moon or the low pressure atmosphere on

the Martian surface. At present there are no materials that

would be generally accepted as sufficiently transparent,

lightweight, and strong enough to meet all of these criteria

at a full earth normal pressure. However, reducing the

pressure within a plant habitat would consequently reduce

the intrinsic strength required for such structures and

materials, reducing the mass of material that must be

lifted from the Earth’s surface, and potentially allowing

the capture of ambient light as a resource.

In this review we present a brief history of the various

atmospheric pressures and gas compositions that have

been used within the human-habitable vehicles of the

space programs, with an eye toward the possible

atmospheric configurations that might be used in future

vehicles and habitats. This narration is followed by a

general discussion of the uses of low pressure

atmospheres in plant biology applications. These two

threads will be integrated with a discussion of

experiments focused specifically on low pressure

atmospheres in plant space biology applications. We will

then develop an argument that low atmospheric pressures

present a serious environmental challenge to plants, a

challenge that requires an adaptive response and

redirection of metabolic resources. Understanding of this

response is enhanced by analysis of the gene expression

Table 1. Atmospheric pressure relative to altitude – a biological perspective. These data provide a perspective of the habitation of humans,

flora and fauna at increasing altitude and reduced atmospheric pressure.



changes that take place as plants respond and adapt. Much

like response and adaptation to other environmental

stresses, such understanding can lead to both a definition

of the current limits of terrestrial plants as well as a path

for producing plants with enhanced capacity for growth

and production at low pressures. Such information is

critical for the space program, as atmospheric pressure

directly impacts the designs and operational procedures

that are being considered for future space vehicles and

extraterrestrial habitats, including greenhouses.

ATMOSPHERIC PRESSURES IN SPACE

EXPLORATION

It is a generally accepted fact that reduction in the

pressure differential between an internal and external

environment correlates with reduction in engineering

costs, especially in the form of structural mass. Given that

mass is such a crucial cost variable in launch

considerations, low atmospheric pressure environments

have been utilized throughout the human space programs.

Lower atmospheric pressures simply reduce the masses of

structural components of space vehicles and have the

associated effect of reducing the amount of atmospheric

consumables required for the mission. Historically such

reductions in mass allowed for increased mission lengths

and increased payload masses.

The Mercury, Gemini, and Apollo environments were

operated at 34 kPa. In order to compensate for the

hypoxia attendant with such a low pressure, the gas

composition of the atmosphere was maintained at 100%

oxygen (Baker, 1981; Martin and McCormick, 1992)

(Table 2). While the pure oxygen environment provided a

suitable mitigation of hypoxia for the astronauts, that

atmosphere carried with it the risk of fire. It is worth

noting that recent studies have also begun to elucidate the

negative effects of prolonged exposure to pure oxygen

environments; however, hyperoxia appears to be toxic

only at partial pressures above 30 kPa. It should also be

noted that all Extravehicular Vehicle Activity (EVA)

space suit environments, beginning in the Gemini

program, have been maintained at 26 kPa in order to

minimize the physical effort required for manipulations of

the suit components. This need to operate EVAs at

reduced atmospheric pressure imposes constraints on

atmospheric management that continue to the present day

(see Table 2). The Skylab environment was also operated

at 34 kPa; however, the composition of the atmosphere

was maintained at 70% O2 : 30% N2. This pressure and

composition was a compromise that reduced the

engineering costs as well as the fire risk, yet maintained

astronaut health with regard to hypoxia. EVAs during

Skylab were conducted with minimal transition to the 26

kPa, pure oxygen environment of the EVA suits, so that

no pressure changes occurred within Skylab during EVA

activities or EVA preparations. Anecdotal evidence

indicates that the Skylab astronauts noticed no obvious ill

effects of living for prolonged periods at hypobaric

pressures, save for the attendant cooling effects of rapid

evaporation of water and sweat and the difficulty of

hearing due to poor sound propagation.

Table 2. US Space vehicles and atmospheric pressure. These data provide descriptions of various NASA orbital and transit vehicles with

respect to their internal cabin atmospheres.



The Space Shuttle, Mir and the International Space

Station environments were and are operated at Earth-

normal pressures near 101 kPa with an Earth-normal gas

mixture of 21% oxygen. Operation at 101 kPa solves

many of the issues of atmospheric pressure and

composition effects on biology. However, there remain

two pressure issues in recent and current operations;

unintentional excursions to low pressures during leaks,

and intentional reductions in pressure to accommodate

EVA activities. Mir suffered several leak events that

seriously impacted operations (Holliman and Aaron,

1997; Zak, 2000), most notably after collision with a

Progress resupply vehicle. While the ISS maintains

pressure in preparation for and during EVAs (requiring

astronauts to do pressure / atmosphere accommodation in

the air lock), the pressure in the Shuttle is lowered to 70

kPa for 24 hrs or more (Figure 2) to accommodate the

acclimation-deacclimation activities that are necessary for

the human transition between 101 kPa / 21% oxygen and

26 kPa / 100% oxygen (Winkler, 1992; Wieland, 1998).

The reduced pressures are employed to facilitate both the

pre-breathing requirements as well as the actual EVA

events.

Figure 2. Pressure profile from the orbiter Endeavour during

the day of the spacewalk EVA on December 10, 2001. The

actual time of the EVA itself was 4 hr 12 min, yet the shuttle

pressure was altered for essentially a full day to accommodate

all of the acclimation for the astronauts to move to and from the

lower pressure, pure oxygen environment of the EVA suits. Data

courtesy of Joe Benjamin, Dynamac, KSC.

PLANTS IN SPACE VEHICLES

Plants have experienced virtually all of the spaceflight

pressure scenarios mentioned above. Seeds were carried

aboard Gemini and Apollo missions, most notably in the

Biostack series of experiments of Apollo and in the Moon

Trees, which were personal effects of astronaut Stu Russa

on Apollo 14. Apparently seeds were also carried by

Astronaut Ed White in his space suit during the first EVA

space walk on Gemini 4. However, in these instances the

exposure was of quiescent seeds, with no studies of those

seeds performed until their return to earth and subsequent

growth at earth normal pressures. Nonetheless, the corn

and Arabidopsis seeds of Biostack germinated and grew,

though with abnormalities generally correlated with hits

of heavy ion radiation. On Skylab, rice plants were

sprouted and grown in a study of tropisms, inadvertently

demonstrating plant germination and development at

reduced atmospheric pressure, within the enhanced

oxygen levels of Skylab (see Table 2 and see also

http://history.nasa.gov/SP-401/ch5.htm). On Mir, plants

were being grown during the Progress collision and

subsequent loss of atmospheric pressure. Perhaps most

importantly, plant experiments have been conducted on

the Space Shuttle on flights where EVA activities

occurred. While many shuttle environmental variables are

replicated in simulation chambers at KSC, atmospheric

pressure is simply not addressed in the simulator ground

controls. Therefore plants have experienced a number of

exposures to spaceflight relevant atmospheric pressures,

but not always in situations where the effects of that

atmospheric pressure might have been noted or

accounted. This concept of unrecognized consequences of

changes in atmospheric pressure extend to several

relevant but non-spaceflight environments, such as the

KC-135 parabolic flight aircraft, which experiences

pressure deltas during each parabola as the plane’s

pressurization system attempts to maintain cabin pressure

during the extreme changes in altitude associated with

parabolic flight (Figure 3).

Figure 3. Pressure profile of a typical KC-135 parabolic flight.

These data were collected by a HOBO datalogger during a

typical life sciences KC-135 flight in January of 2000. These

data are not calibrated and are presented for demonstration

only. Note the pressure drop as the plane takes off and the

pressure stabilizes at a lower pressure characteristic of airliners

at cruise altitude. Note also that each parabola is characterized

by a further transient drop in pressure as the aircraft cabin

pressure system struggles to maintain a constant pressure. The

parabolas occur usually in groups of ten, characterized by the

closely spaced deviations in cabin pressure. The stable areas of

the graph between the groups of parabola pressure deviations

denote the times of constant altitude turns during which the KC-

135 changes direction to orient for the next series of parabolas.



PLANT PHYSIOLOGY IN HYPOBARIC

ENVIRONMENTS

Although plants are able to survive and even thrive at

pressures well below the threshold of typical human

habitation, it is very likely that most plants would

perceive such hypobaria as a stressful environment

requiring response and adaptation. The likelihood of a

hypobaria stress response is derived from knowledge of

plant responses to other forms of environmental stress,

including hypoxia, cold and dehydration (Ferl et al.,

2002). Yet, since hypobaria, alone, is not a native

terrestrial environment, the nature of the hypobaria stress

response could be entirely unique. Thus, fundamental

understanding of plant responses and adaptability to low

atmospheric pressures is a requisite part of developing

insights into pressure effects on terrestrial life-forms in

general as well as maximizing plant growth under

hypobaric advanced life support conditions. There is a

rich history of low atmospheric pressure research within

the plant biology community, a history that explores the

responses of plants to hypobaria and the adaptations of

plants to related environmental stresses such as hypoxia.

Most early and many recent hypobaria observations were

focused on direct physiological changes and metabolic

impact of low atmospheric pressures on plants (reviewed:

Daunicht and Brinkjans, 1996; Salisbury, 1999; Wheeler

et al., 2001; Corey et al., 2002; Ferl et al., 2002). While

many of the early investigations had a connection to the

international Advanced Life Support (ALS) community,

others explored the effects of low pressure environments

on the post-harvest physiology of fruits, vegetables and

flowers in commercial applications (e.g. Burg, 2004 and

references therein).

One of the earliest hypobaria studies attempted to recreate

a Mars-like atmosphere in bell jars and observed the

effect of that environment on rye seed germination.

Although seeds would germinate in an approximation of

martian atmospheric composition (0.24% CO2, 0.09% O2,

1.39% argon, balance of N2) at normal and slightly

reduced atmospheric pressures (101 kPa and 50 kPa),

lower ranges of pressure (10 kPa and 3 kPa) would not

support germination (Siegel et al., 1962, 1963). Another

early study was conducted by the Air force in 1968 to

explore the utility of using higher plants for food and

atmospheric regeneration in extended human space

missions (Mansell et al., 1968). Since the atmospheres of

the Mercury, Gemini and Apollo spacecraft were kept at

lower atmospheric pressures with elevated partial

pressures of oxygen, it was important to determine

whether suitable plants could also thrive in similar and

related hypobaric conditions. The growth and

development of turnip plants (Brassica rapa) were

evaluated at 50 kPa compared against a control of 93 kPa.

In these experiments, the partial pressure of oxygen was

kept at normoxic conditions of 21 kPa. After 21 days of

growth, it was concluded that there were no adverse

effects seen in the 50 kPa plants, although it was noted

that transpiration rates were elevated compared to the 93

kPa control (Mansell et al., 1968). It is interesting to note

in a historical context how early the spaceflight

community was actively considering the utility of plants

as part of advanced life support systems, under space-

vehicle relevant pressures and gas compositions. These

plant experiments were performed shortly after a series of

animal experiments were conducted in 1965 to determine

the toxicology of the 34 kPa low pressure, pure oxygen

environment being employed for the space capsule

atmosphere (Thomas, 1965). A few years later plants

were also subjected to germination and growth

experiments in a simulated space capsule environment

(Lind, 1971). It can be concluded that these early studies

clearly established the viability of both plants and animals

in low atmospheric pressures and within gas compositions

relevant to space exploration.

In the 1960’s it was also discovered that hypobaric

pressures may have an impact on plant hormone related

physiology, and this realization expanded the interest in

hypobaric research to the commercial fruit industry.

Holding fruit at sub-atmospheric pressures delayed

ripening (Burg and Burg, 1965) and subsequent studies

related this phenomenon to the depletion of ethylene from

fruit tissues that was accelerated by the hypobaric

conditions. Tomatoes, bananas, mangos, cherries, limes

and guavas were incubated in atmospheric pressures

ranging from 48 kPa to 20 kPa and compared to similar

treatments in normal atmospheric pressure (Burg and

Burg, 1966a, 1966b). Later studies used hypobaric

conditions to enable the discrimination between the

effects of ethylene and abscissic acid in the formation of

abscission zones. Bell jars with atmospheres reduced to

20 kPa were used to determine that ethylene is the

primary effector of abscission in citrus fruits (Cooper and

Horanic, 1973). Since these early studies there has been a

wide application of these principles (e.g. Dilley et al.,

1975; Spalding and Reeder, 1976; Lougheed et al., 1978;

Nilsen and Hodges, 1983; Jardine et al., 1984; Kirk et al.,

1986) and this field has been reviewed recently (Burg,

2004).

Investigations into the underlying physiology of growing

plants in hypobaric environments continued to be

explored with both basic physiology and spaceflight

applications in mind, although early experiments

sometimes produced mixed results. For instance, in one

study tomato seedlings grown at 17 kPa were stunted in

growth, while plants grown at 33 kPa were more robust

when compared to the 100 kPa control (Rule and Staby,

1981). Yet in another, tomato plants were uniformly

stunted in reduced atmospheric environments of 40 and

70 kPa compared to 100 kPa control (Daunicht and

Brinkjans, 1992) while the negative effects of extreme

hypobaria (3 kPa) on rye seed germination could be

mitigated with added oxygen (Andre and Richaux, 1986).

Thus, as plant growth experiments explored low pressure

environments, it was found that the composition of the

air, especially with respect to O2 and CO2 can have a

profound effect on the physiological response of the

plants growing in a reduced overall pressure,

complicating the interpretation of the underlying effects



of low pressure that are independent of gas composition.

The two atmospheric components that contribute most to

complicating a purely hypobaric response are CO2 and O2.

For CO2 related processes, the increase in molecular

diffusion rates of CO2 at lower atmospheric pressures

enhances the ability of plants to take up the gas in

hypobaric environments (Gale, 1972). Thus the decrease

in absolute CO2 concentrations at lower pressures is

counterbalanced by the increase in CO2 diffusion rates. In

natural environments this positive effect could in turn be

counteracted by the decreases in temperature that

accompany reduced atmospheric pressures at high

altitudes (Gale, 1973), as well as by the concomitant

decreases in stomatal aperture, increases in transpiration

rates, and the increases in water exchange demands that

accompany higher elevations and lower pressures (Smith

and Geller, 1979; Kirk et al., 1986; Mott and Parkhurst,

1991; Gale, 2004). It has been suggested that such

demands might impact stomatal distribution and density

in plants adapted to high altitudes (Körner et al., 1986)

and this correlation has even been used to predict

probable attitudes of habitation for fossilized leaves

(McElwain, 2004). However, there is some dispute in this

latter application (Johnson et al., 2005) as stomatal

distribution can be associated with changes in CO2

concentrations independent of altitude (Lake et al., 2001).

In experimental scenarios where temperature and pressure

can be controlled, it appears that, at least for hypobaric

pressures of about 50 kPa and above, the increase in CO2

uptake facilitated by increased diffusion rates of CO2

counterbalances the relative scarcity of the gas (Smith and

Donahue, 1991).

As mentioned in earlier sections, the amendment of

oxygen to hypoxic atmospheres can have a profound

effect on the ability of animals to cope with hypoxic

environments. Oxygen also ameliorates some of the

effects of hypobaric stress in plants. One such study

demonstrated that wheat was capable of germinating and

growing at total atmospheric pressures of 10 kPa, and that

pressures of 20 kPa even enhanced growth when the

partial pressure of nitrogen was kept low by using oxygen

to displace much of the nitrogen in the maintenance of the

total atmospheric pressure. The effect of this replacement

created an atmosphere with a total pressure of 20 kPa, and

partial pressures of O2 at 14 kPa, N2 at 3.4 kPa and CO2 at

3.4 kPa. Interestingly, the positive effects of this

atmosphere on growth were couched in terms of a

reduction of nitrogen rather than an increase of oxygen

(Andre and Massimino, 1992). Indeed, if a pure oxygen

atmosphere is used, rye seeds are capable of germinating

at pressures of 3 kPa (Andre and Richaux, 1986).

However, oxygen does not ameliorate all issues of growth

and development at low pressures. In a study with mung

bean seedlings that compared mitochondrial respiration

rates with overall growth, it was found that while

respiration responded to oxygen concentration

independently of the overall atmospheric pressure down

to 21 kPa, quite the opposite was found for seedling

growth. In this case, growth (as assayed by mass

accumulation and length of seedlings) was negatively

correlated with a decrease in pressure and was

independent of the partial pressure of oxygen (Musgrave

et al., 1988a). Arabidopsis and rice also appear to be

impacted by oxygen concentrations in their ability to cope

with hypobaric conditions. Although both species could

grow at atmospheric conditions of 25 or 50 kPa of total

pressures, the addition of oxygen to a partial pressure of

at least 10 kPa appeared to compensate for any

disadvantageous of growing in a hypobaric environment

(Goto et al., 2002).

Independent of the gas composition in a hypobaric

environment is the more direct effect that low

atmospheric pressures have physically on gas relations in

plant physiology; all other things remaining constant, if

the atmospheric pressure is reduced, the rate of gas

exchange will increase. Evaporation, for example,

increases as pressure is reduced, an effect that can account

at least in part for increased plant transpiration at low

pressures (Rygalov et al., 2002). However, it is not clear

whether all things do remain constant in a plant adapting

to hypobaric conditions. In a short term experiment CO2

assimilation rates and transpiration rates were enhanced

for spinach in 25 kPa environments compared to 101 kPa

controls. However, in long term experiments it was

demonstrated that the gas exchange rates for plants in 25

kPa did not vary from those grown at normal pressures.

One apparent reason for this result was that over time, the

stomatal openings in these plants became correspondingly

smaller, thereby reducing the rate of gas exchange

through these pores (Iwabuchi and Kurata, 2003). Long

term experiments with lettuce at slightly reduced

atmospheric pressure (70 kPa) demonstrated that plants

could adapt with no adverse effects, and even tended to be

slightly more robust than their 101 kPa counterparts

(Spanarkel and Drew, 2002).

These findings suggest that plants appear to respond to

low atmospheric pressures through a fairly complex set of

adaptations, and further, there are situations where the

benefits of hypobaric environments may outweigh the

metabolic cost, in part through effects on the gaseous

plant hormone ethylene. Where plants are cultivated in

closed containers, hypobaric environments of 30 kPa total

pressure appear to offset the detrimental effects of

ethylene and other volatile biological compounds that

normally accumulate in closed systems (He et al., 2003).

Some of these effects appeared to be due to an inhibition

of ethylene production by the low partial pressure of

oxygen, as hypoxia alone was not as uniformly effective

in ethylene management. Although ethylene production

could be inhibited in lettuce grown at 101 kPa with a

partial pressure of oxygen equivalent to that found at 30

kPa of total pressure, the same was not true of wheat.

Ethylene production in wheat could only be controlled

when the overall atmospheric pressure was reduced to 30

kPa (He et al., 2003).



LOW ATMOSPHERIC PRESSURE HABITATS

From a physiological point of view, plants cope well with

hypobaric atmospheres, and conditions can be engineered

for closed system environments where plants may even

benefit from atmospheric pressures as low as 30 kPa. This

set of circumstances lends itself very well to the physical

engineering needs of vehicles and habitats that

exploration may take to other planetary surfaces (Wheeler

et al., 2001; Corey et al., 2002) and sets the stage for the

genetic engineering to further enhance the production of

plants that may populate such habitats (Ferl et al., 2002).

Low atmospheric environments were proposed for

Martian greenhouses in early concepts that examined

human exploration on Mars (Boston, 1981). It was

thought that low pressure greenhouses would be the most

effective means to grow plants in support of a mission on

a planet that had a vast differential between the external

atmospheres (ca. 0.7 kPa) and the internal environment.

Early experiments to support the concept employed plants

grown in largely CO2 atmospheres at a total pressure of 5

kPa. Radish, alfalfa and mung beans were germinated and

grown for 2 to 4 days with only minimal mortality (10%)

and much of the mortality was due to secondary effects,

such as fungal contamination (Boston, 1981). The idea of

an inflatable structure within a man-made or natural (e.g.

lava tube) rigid structure captured many imaginations and

has endured as a valid model for more than 20 years. The

inflatable greenhouse concept supports the notion that on

a long duration mission to Mars resupply is not an easy

operational option, and any decrease in initial supply

mass enhances launch capabilities (McKay and Toon,

1991; Schwartzkopf and Mancinelli, 1991; Mitchell,

1994; Schwartzkopf, 1997; Kennedy, 1999; Salisbury,

1999; Clawson, 2000; Fowler et al., 2000; Alling et al.,

2002; Corey et al., 2002; Sadler and Giacomelli, 2002;

Hublitz et al., 2004). Habitats have been envisioned that

range from significantly reduced atmospheric pressure

that would require pressure suits and robotic interventions

(Boston, 1981; Clawson, 2000; Corey et al., 2002) to

habitats that are on the edge of human comfort (50 kPa)

that would permit short excursions by crew with minimal

support (Hublitz et al., 2004). Many of these designs

employ transparent inflatable structures to make maximal

use of ambient light (Clawson et al., 2005).

PLANT GENE EXPRESSION IN HYPOBARIC

ENVIRONMENTS

Plants are able to cope with a vast variety of

environmental conditions that differ from a well hydrated

temperate meadow. Plants can engage metabolisms that

enable them to thrive in niches characterized by extremes

in temperature, humidity and oxygen availability. The

responses associated with these stress environments (cold,

desiccation, flooding, heat shock) have been well

characterized; yet until recently, it was not known

whether the response strategies elicited by hypobaria

engaged unique pathways or similar pathways as the

known environmental stresses (Ferl et al., 2002). To

address this question, experiments were conducted with

Arabidopsis to examine the patterns of gene expression on

a genome wide scale as plants were introduced to

hypobaric and comparable hypoxic environments (Paul et

al 2004). Affymetrix GeneChip

8K arrays were used to

characterize the effects of 24 hour exposure of

Arabidopsis seedlings to an atmospheric environment of

10 kPa. The gene expression patterns were then compared

to those of plants exposed to a normal pressure hypoxic

environment of 2% oxygen (the partial pressure of O2 at

10 kPa) and both patterns compared to 24 hour exposure

to Earth-normal air at 101 kPa.

The hypobaric and hypoxic environments were created

within a Low Pressure Growth Chamber (LPGC). The

LPGC controlled lighting, temperature, CO2 partial

pressure and humidity across all experiments such that the

only variables were either atmospheric pressure or oxygen

content. The experimental plants were nine day old

Arabidopsis seedlings grown on nutrient agar plates, a

configuration that ensured that both roots and shoot

received identical atmospheric conditions throughout the

treatment, and that the entire surface of the plant received

the full impact of the environment in which it was placed.

It is also important to note that the relative humidity of

the plant’s microenvironment inside the culture plate

remained at or above 95% in every treatment. Further, all

plants remained turgid and showed no signs of wilting or

desiccation at the end of the 24 hours of treatment.

Of the 8,000 genes represented on the array, more than

200 were differentially expressed in the shoots of plants

exposed to a hypobaric environment of 10 kPa for 24

hours. A comparable number of genes were similarly

differentially expressed in hypoxic treatment - yet only a

fraction of the two sets of differentially expressed genes

overlapped in patterns of expression (Figure 4). Many of

the hallmarks of hypoxic stress were found in both the 10

kPa and 2% O2 treatment, as oxygen is limiting in both

scenarios (groups 3 and 4, as well as members of groups 2

and 5, Figure 4). Genes required to support fermentative

pathways and those involving oxygen transport are

required under low oxygen conditions (e.g. Klok et al.,

2002) so it is not surprising to find representatives of

these metabolisms in both treatments. The genes

repressed by both hypoxia and hypobaria included

examples from a wide range of metabolic pathways that

are globally down-regulated in response to hypoxic stress

(Sachs et al., 1980; Sachs et al., 1996; Vartapetian and

Jackson, 1997; Klok et al., 2002). The hypobaria response

therefore included many of the gene activity changes

typical for a hypoxic response, but the hypobaric response

was not limited to genes involved in hypoxia.

The group of genes that was most highly induced by

hypobaria, yet unaffected by hypoxia, were ones involved

with desiccation-related metabolic pathways. Many of the

genes represented in this group displayed as much as a

30-fold difference in expression between 2% O2 and 10

kPa (see group 1, containing 30 genes, Figure 4). Among

the most abundant genes in this category were cold



induced genes (e.g. Cor28), dehydrins, which are

typically associated with ABA regulated seed storage

proteins (e.g. LEA) as well as many other examples of

desiccation and ABA regulated genes (Yamaguchi-

Shinozaki and Shinozaki, 1993a; Siddiqui et al., 1998;

Thomashow, 1999; Finkelstein et al., 2002; Soulages et

al., 2003). Also found in this category were genes that are

associated with the regulation and distribution of stomates

in Arabidopsis leaves (Berger and Altmann, 2000) and

many genes associated with the mediation of signal

transduction, especially those related to kinases (Guo et

al., 2002; Yoshida et al., 2002), calcium binding

(Takahashi et al., 2000; Sadiqov et al., 2002) and a

cytochrome p450 (Reddy et al., 2002). There were two

smaller groups of genes that exhibited interestingly

unique patterns of gene expression among the hypoxic

and hypobaric treatments. First, there were a few genes

that were repressed in hypoxia, but were unaffected by

hypobaria. Representatives from this group encoded

proteins similar to those involved in oxygen sensing

processes that are mediated through a heme- or iron-based

sensor (Aravind and Koonin, 2001; Zhu and Bunn, 2001;

Quinn et al., 2002). Second, there were those that were

induced in hypoxic environments, while remaining

unaffected by hypobaria (some of the genes in group 6 of

Figure 4). Genes encoding heme-related proteins were

also found in this group, as well as genes typical of a

hypoxic stress response. The dissimilarity in gene

expression patterns in what might be considered related

oxygen sensing systems, suggests that that hypobaria and

hypoxia may differentially activate and repress certain

alternative oxygen sensing and transport pathways.

Figure 4. Differential expression of Arabidopsis genes in response to hypobaria (10 kPa) and hypoxia (2% O2). Over 200 genes of the

8,000 of the Affymetrix Arabidopsis Gene Chip© were differentially expressed in the shoots of plants exposed to a hypobaric environment of

10 kPa or a hypoxic treatment of 2% O2 for 24 hours. The panel on the left contains three columns of stacked, colored lines. Each line

represents one gene that exhibits differential expression with regard to the Earth Normal (101 kPa / 21% O2) control. When a gene is

induced relative to the control it is indicated in red, when it is repressed it is indicated in green. The reference column of normal sea level

pressure looks entirely black as all expression is calculated relative to the values therein. The two columns to the right (hypoxia and

hypobaria) display patterns of differential gene expression and are clustered into 7 groups of gene exhibiting similar response. The cluster

number and the number of genes represented in that cluster are indicated between the two panels. The graph on the right displays the

relative normalized gene expression averages (Paul et al., 2004) for the groups of genes within the group for hypoxic (open bars) and

hypobaric (closed bars) treatments. The color version of this figure can be found on line: http://asgsb.org/publications.html ). In the

grayscale print version, induced appears as a darker gray and repressed as a lighter gray.



The specificity of the hypobaric response was also

evaluated in transgenic Arabidopsis plants transformed

with Adh/GFP or Cor78/GFP transcriptional promoter

fusions. Induction of the alcohol dehydrogenase gene

(Adh) is a well characterized component of a hypoxic

stress response while the cold response 78 gene (Cor78)

is typically associated with cold and drought stress, and in

these experiments, hypobaria. The sensing portions of the

transgenes were composed of the promoter regions of

these genes. The structural portion of each transgene

encodes Green Fluorescent Protein (GFP), which can be

visualized non-destructively in vivo when illuminated

with short wave blue light (488 nm). The plants were

exposed to hypobaric and hypoxic environments for 24

hours and then illuminated with 488 nm light to determine

the extent of tissue specific reporter gene expression. The

Adh/GFP plants showed abundant reporter gene

expression in the meristematic regions of the shoots and

roots with both hypoxic and hypobaric treatment,

however, the Cor78/GFP plants only expressed the

reporter gene in response to hypobaric treatment

(Figure 5). In addition, although Adh/GFP and

Cor78/GFP both showed reporter gene expression in

hypobaria, the tissue-specific patterns of expression were

different for each plant. Adh/GFP expression was

concentrated in the apical meristem and newly emerged

leaves, particularly in trichome cells. Very little

expression of the reporter was seen in more mature tissues

in Adh/GFP plants. Cor78/GFP on the other hand, was

expressed throughout the leaves, although again

predominated in the newly emerged leaves and trichome

cells (the latter partly owing to the large, clear nature of

these cells). In addition, GFP expression extended from

the aerial portion of Cor78/GFP plants along the stem to

the root/shoot junction (Paul et al., 2004). Thus, the

response to hypobaria is not limited to those tissues

responding to hypoxia, and that the signaling mechanisms

for the hypobaria-specific response are fundamentally

different from those of the hypoxia response with regard

to the distribution of those cells undergoing the

response.The extensive changes in gene expression

patterns clearly indicate that adaptation to low pressure

environments requires a robust and dynamic adaptive

response. Some of the altered gene expression patterns are

similar to those involved in the hypoxic stress response,

as would be expected from the low oxygen partial

pressures present in hypobaria; however, many of the

changes in gene expression patterns are unique to

hypobaria. These unique differential gene expression

patterns demonstrate that hypobaria is not at all equivalent

to hypoxia as an abiotic stress and clearly suggest that, for

plants, the response to hypobaria is much more complex

than the acclimation to the reduced partial pressures of

oxygen inherent to low atmospheric pressures. These

conclusions regarding complexity based on molecular

data begin to explain why the physiological data in

previous experiments do not necessarily yield easy

explanations.

So, if hypobaria is a more complex stress than hypoxia,

what other metabolic processes does it impact? As

indicated by the induction of the Cor78 gene discussed

above, one of the largest group of genes that showed over

expression in hypobaria while being unaffected by

hypoxia is characterized by genes associated with

desiccation and ABA signaling related processes (e.g.

Ozturk et al., 2002). Examples include ABI2 (Chak et al.,

2000), the APL3 subunit of ADP-glucose

pyrophosphorylase (Weber et al., 1995; Rook et al.,

2001), LEAs, (e.g. Siddiqui et al., 1998; Kim et al., 2002)

Figure 5. Tissue specific transgene expression in response to hypobaria (10 kPa) and hypoxia (2% O2). Transgenic plants containing

either the Adh/GFP or Cor78/GFP transgene were photographed in white and 488nm fluorescent light. The top row shows Adh/GFP

plants after exposure to 24 hours of 10 kPa hypobaria (left) and after 24 hours of 2% O2 hypoxia (right). The apical meristems regions and

the roots (where visible) are expressing the GFP reporter, and these regions glow green in the right hand picture of each pair. The bottom

row shows Cor78/GFP plants after exposure to 24 hours of hypobaria or hypoxia. In the case of the Cor78/GFP plants, transgene

expression was induced solely by the hypobaric treatment (left hand panels), plants exposed to hypoxic environment did not express the

transgene (right hand panels). The color version of this figure can be found on line: http://asgsb.org/publications.html ). In the grayscale

print version, GFP expression appears as white or light gray on the black background of the photographs.



dehydrins and rab-like proteins, (e.g. Mantyla et al., 1995;

Nylander et al., 2001; Kizis and Pages, 2002) rd29B and

cor15B (Wilhelm and Thomashow, 1993; Yamaguchi-

Shinozaki and Shinozaki, 1993b) and aldehyde

dehydrogenase (Kirch et al., 2001). Thus, it appears that a

primary impact of a hypobaric environment, independent

of oxygen stress, is on the mechanisms by which a plant

perceives and responds to water movement or desiccation.

It is important to emphasize that it is the perception

mechanism that is being impacted, not the actual

desiccation process. The plants in these experiments were

grown in a humid environment (>95% rh within the

plates), showed no loss of fresh weight or turgor, and yet

still responded to 10 kPa as if they were dehydrated or in

the process of dehydration. This pivotal result indicates

that the desiccation response is likely due to the

perception of increased water flux caused by the low

pressure environment rather than actual loss of water

content within the plants.

Thus the analysis of the differential gene expression

patterns from these experiments lead to two fundamental

conclusions: first, that hypobaria does not equal hypoxia,

and second, that the primary metabolic pathways that are

engaged by hypobaric stress include those that encompass

the desiccation response or otherwise touch ABA

mediated metabolisms. The response to hypobaria is

much more complex than the acclimation to low partial

pressures of oxygen. Therefore, it would be predicted that

complete compensation for hypobaria in plants would not

be accomplished by increasing the oxygen content, as was

done for low pressure environments of the Mercury,

Gemini and Apollo vehicles. The desiccation-related

response of Arabidopsis plants at 10 kPa in the absence of

real desiccation or wilting also raises the question as to

whether adaptation to a low atmospheric pressure actually

requires the activation of desiccation related pathways, or

if the induction of desiccation-related metabolism may

not be necessary for survival. If the desiccation response

is not necessary for adaptation to low pressure, then the

inappropriate induction of these pathways could represent

costs to production and genetic engineering to remove the

response may be beneficial. If the desiccation response is

indeed required, then enhancing the desiccation response

may increase production at low atmospheric pressure.

CONCLUSION

The experiments and conclusions discussed here expose

the need for continued evaluation of the effects of low

pressure on biological systems. In some ways, the gene

expression data suggest that we actually have only a small

fraction of the data necessary to make informed choices

on the gas composition and pressures within vessels that

support plant growth. By extension, these data also beg

the questions of what sort of undetected responses and

adaptations to low pressures occur in other organisms,

including humans, and whether low pressure

environments have contributed to any of the existing

spaceflight data on biology and biological responses in

spaceflight environments. There is little data available on

the response of any animal system to hypobaric

environments at the level of gene expression, but there are

studies that explore the physiological responses of

animals to hypobaric environments. Of particular interest

are those that indicate hypobaric environments could

contribute to compromised health and resistance to

disease. Early ground-based studies in mice demonstrated

that hypobaric environments, even when supplemented

with oxygen, can lead to stimulated growth of

gastrointestinal bacteria (Gillmore and Gordon, 1975),

retard healing of staphylococcus skin infections (Schmidt

et al., 1967), increase mortality in animals with

intraperitoneal infections (Ball and Schmidt, 1968) and

depress interferon levels (Huang and Gordon, 1968).

More recently, studies have linked hypobaric

environments with an impaired immune response in rats

(SaiRam et al., 1998) mice (Biselli et al., 1991) and

humans (Meehan et al., 1988; Facco et al., 2005). Rarely

has hypobaria been studied independently from hypoxia,

but a recent study examining the effects of mild hypobaria

on fluid balance issues conducted hypobaric treatments in

normoxic atmospheres to control for the effects of

hypoxia. They concluded that although there is a

synergistic effect of hypoxic and hypobaric stress at

altitude, hypobaria alone can adversely impact the fluid

and ionic balance of humans (Loeppky et al., 2005).

So the short term questions surrounding low pressure

spaceflight and extraterrestrial environments involve

deepening our understanding of responses and adaptation

to hypobaria for all organisms and biological systems that

may be a part of spaceflight and extraterrestrial habitats.

Indeed, unpublished molecular data indicate that even the

relatively small pressure drops to 75 kPa that occur in the

space shuttle effect the expression of hundreds of genes in

Arabidopsis. That being the case, even seemingly small

choices about operations and procedures conducted within

habitat environments can have measurable and dramatic

impacts on the biology of those habitats. Therefore very

near term decisions involving, for example, the choices of

atmospheric composition and pressure in the Crew

Exploration Vehicle will have long term implications for

biology. In the longer term, the use of low pressure

environments may enable life support systems that

otherwise would be impossible to construct or maintain

under higher atmospheric pressures, keeping viable the

concepts of inflatable greenhouses. For example,

transparent greenhouses could be erected on Mars using

currently available materials, but only if the internal

pressure of the greenhouse could be maintained below 7.5

kPa (Boston, 1981). The rather dramatic changes in gene

expression that occur at 10 kPa suggest that response and

adaptation to lower pressures will be even more

extensive, but the study of such pressures are well within

the capacity of current experiment chamber design and

molecular expression technology.



ACKNOWLEDGEMENTS

The spaceflight and low pressure experiments of the Ferl

laboratory were supported by NASA grants NAG 10-316,

NAG 10-291 and AO-99-HEDS-01-032. The authors

wish to recognize the tremendous contributions of the

many scientists whose work is summarized in this review.

In particular, we thank Andrew Schuerger, Jeff Richards,

Ken Corey, Ray Bucklin, Ray Wheeler, Vadim Rygolov,

Phil Fowler and other folks involved in developing the

low pressure facilities at KSC. We thank Mike Dixon and

his associates at the University of Guelph and the

Controlled Environment Systems Research Facility

(CESRF). We thank Bill Wells, Joe Benjamin and Kelly

Norwood from KSC for providing source material

regarding shuttle and KC-135 atmospheric pressures. We

also thank our colleagues at UF who participated in low

pressure and recent KC-135 experiments, including

Jordan Barney, Matt Reyes, Bill Gurley, Mike Manak and

John Mayfield.

The authors also wish to acknowledge the difficulty of

deriving original references for some of the discussions

presented in this paper. Any mistakes or

misrepresentations or misattributions are regretted and we

would welcome any comments, corrections or any

additional references at [email protected] or [email protected].

This paper is dedicated to the memory of Guy Etheridge.

His appreciation of science and love of space research had

such a wonderful influence on all.

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