Table Of Contentv
Contents
FOREWORD OFTHESERIES EDITOR TOVOLUME9.. .. .. .. .. .. .. . .. .. vii
AugustoCogoli
DEVELOPMENTAL BIOLOGY RESEARCH IN SPACE:
INTRODUCTORY REMARKS OFTHEVOLUME EDITOR .. .. .. .. . .. .. ix
Hans-Ju¨rg Marthy
PLANT REPRODUCTIVE DEVELOPMENT DURING
SPACEFLIGHT. .. .. .. .. .. .. .. . .. .. .. .. .. .. . .. .. .. .. .. .. .. . .. .. . 1
MaryE. Musgrave andAnxiuKuang
BDELLOID ROTIFERS AS MODEL SYSTEM TOSTUDY
DEVELOPMENTAL BIOLOGY IN SPACE. .. .. .. . .. .. .. .. .. .. .. . .. .. 25
ClaudiaRicci andChiaraBoschetti
DROSOPHILA MELANOGASTER AND THEFUTURE OF
EVO-DEVO BIOLOGY IN SPACE.CHALLENGES AND
PROBLEMS IN THEPATH OFAN EVENTUAL
COLONIZATION PROJECT OUTSIDE THE EARTH.. .. .. .. .. .. .. . .. .. 41
RobertoMarco, DavidHusson, RaulHerranz, Jesu´sMateos and
F.Javier Medina
MORPHOGENESIS AND GRAVITYIN A WHOLE
AMPHIBIAN EMBRYO AND INISOLATED BLASTOMERES
OFSEAURCHINS .. .. .. .. .. .. . .. .. .. .. .. .. . .. .. .. .. .. .. .. . .. .. 83
AkemiIzumi-Kurotani andMasatoKiyomoto
DEVELOPMENTAL BIOLOGY OFURODELE AMPHIBIANS IN
MICROGRAVITY CONDITIONS . . .. .. .. .. .. .. . .. .. .. .. .. .. .. . .. . 101
Christian Dournon
THE DEVELOPMENT OFGRAVITY SENSORYSYSTEMS
DURING PERIODS OFALTERED GRAVITY DEPENDENT
SENSORY INPUT. .. .. .. .. .. .. . .. .. .. .. .. .. . .. .. .. .. .. .. .. . .. . 133
EberhardR. Horn
vi
NEUROPHYSIOLOGY OFDEVELOPING FISHAT
ALTERED GRAVITY: BACKGROUND—FACTS—PERSPECTIVES. . .. .. . 173
Ralf H. Anken
LIFE-CYCLE EXPERIMENTS OFMEDAKA FISHABOARD
THE INTERNATIONAL SPACESTATION. .. .. . .. .. .. .. .. .. .. . .. .. . 201
Kenichi Ijiri
MAMMALIAN DEVELOPMENT IN SPACE .. .. .. . .. .. .. .. .. .. .. . .. .. . 217
April E. Ronca
NEW FACILITIES AND INSTRUMENTS FOR
DEVELOPMENTAL BIOLOGY RESEARCH INSPACE.. .. .. .. .. . .. .. . 253
EnnoBrinckmann
LIST OFMAINAUTHORS .. .. . .. .. .. .. .. .. .. . .. .. .. .. .. .. .. . .. .. . 281
Developmental Biology Research inSpace
H.-J.Marthy (editor) 1
(cid:1) 2003Elsevier ScienceB.V. Allrights reserved
Plant Reproductive Development during Spaceflight
Mary E. Musgrave1,* and Anxiu Kuang2
1Department ofPlant Science, University ofConnecticut, 1376Storrs Road,Unit
4067, Storrs, CT 06269, USA
2Department of Biology, University of Texas Pan American, Edinburg,
TX 78539, USA
Abstract
Reproductivedevelopmentinmicrogravityhasnowbeenstudiedinavarietyof
plants; Arabidopsis, Brassica, and Triticum have been especially well studied.
Earlier indications that gravity might be required for some stage of
reproductive developmenthave now been refuted. Nevertheless,the spaceflight
environmentpresentsmanyuniquechallengesthathaveoftencompromisedthe
ability of plants to reproduce. These include limitations in hardware design to
compensate for the unique environmental characteristics of microgravity,
especially absence of convective air movement. Pollen development has been
shown to be sensitive to high concentrations of ethylene prevailing on various
orbital platforms. Barring these gross environmental problems, androecium
andgynoeciumdevelopmentoccurnormallyinmicrogravity,inthatfunctional
propagules are produced. Nonetheless, qualitative changes in anther and pistil
development have been shown, and significant qualitative changes occur in
storage reserve deposition during seed development. Apart from the intrinsic
biological importance of these results, consequences ofdiminished seed quality
when plants are grown in the absence of gravity will detract from the utility of
plant-basedlifesupportsystems.Byunderstandinggravity’sroleindetermining
the microenvironments that prevail during reproductive development,counter-
measures to these obstacles can be found, while at the same time providing
basic knowledge that will have broader agricultural significance.
Introduction
Although plants have been included in the biological payloads of orbital
platformsfordecades,weareonlybeginningtounderstandhowtheabsenceof
*E-mail:[email protected]
2
gravity impacts long-duration growth and reproductive development. Because
of the well-known tropic responses of plant organs to gravity, the utility of the
microgravity environment for studying signal transduction in plants has been
appreciated (Antonsen and Johnsson, 1998; Kiss et al., 2000; Perbal and
Drissecole, 1994; Volkmann et al., 1986). However, as the Soviets tried to use
plants to supplement the psychological environment of space craft, as well as
give variety and fresh food to astronauts engaged in long-duration missions, it
becameclearthattheabsenceofgravityposedproblemsforplantreproductive
development (Nechitailo and Mashinsky, 1993).
This review will discuss the obstacles to plant reproductive development in
microgravity. Historically, these have included hardware design issues, since
early plant hardware design did not provide an adequate environment to
support plant growth, and plants died at the transition from vegetative to
reproductive development (Halstead and Dutcher, 1984, 1987; Nechitailo and
Mashinsky,1993).Inmorerecentstudies,betterhardwaredesignhaspermitted
an examination of developmental obstacles to reproduction imposed by
microgravity. Reproduction is a complex developmental process. Not only
must the vegetative portionof the plant be sufficiently vigorous to support this
energeticallydemandingprocess,butallofthecomponentsoftheprocessmust
function well. Megasporogenesis, microsporogenesis, pollination, pollen tube
growth,fertilization,embryodevelopment,andseedmaturationareallcomplex
events. This review will show that while none of these processes is absolutely
dependentongravityforitscompletion,environmentalconstraintsimposedby
microgravity have been implicated in qualitative changes in these processes.
Hardware for reproduction studies
More than 10 different types of plant hardware have been used in the
microgravity environment to study some aspect of plant reproductive
development (Table 1). On Kosmos 1129, a simple Plexiglas beaker containing
moist soil had been taken to orbit containing flowering Arabidopsis plants,
whichsubsequentlyproducedseedswhileinmicrogravity(albeitwithonly55%
fertility; Parfenov and Abramova, 1981). On Salyut 6, two open plant growth
chambers,Oasis,andMalachite,wereusedtogrowgardenpeaandEpidendrum
orchid, respectively (Nechitailo and Mashinsky, 1993). In the case of peas
growing in the Oasis module, plants were started from seeds in microgravity,
but did not develop into flowering plants. Similarly, orchids taken to Salyut in
flowerintheMalachitehardwareproducednoadditionalflowerswhileonorbit.
BuildingonthesuccessofworkwiththesmallplantArabidopsis,Ukrainian
scientists tried to grow it in a hermetically sealed hardware called Svetoblok
(Fig. 1a). Designed to clip under a light on the Salyut 6 station, Svetoblok
provided a low light environment for Arabidopsis plants growing on an agar
medium. Plants were developmentally delayed, but eventually flowered
although the androecium and gynoecium aborted (Kordyum et al., 1983).
Table1
Experimentsonplantreproductionduringspaceflighthaveutilizedavarietyofplantmaterialsandgrowthchambers.Theresultsoftheseexperimentshave
beenreviewedelsewhere(HalsteadandDutcher,1984,1987;Musgraveetal.,1997;NechitailoandMashinsky,1993)
Startingmaterial Chamber Ventilation Flowers Seeds Reference
Plantsorseedlings
Arabidopsis (Kosmos1129)a Open þ þ ParfenovandAbramova,1981
PGUc(STS-54) Closed þ (cid:1) Kuangetal.,1995
PGU(STS-51) " þ CO þ (cid:1) Kuangetal.,1996a
2
PGU(STS-68) active þ þ Kuangetal.,1996b
Brassica PGFb(STS-87) active þ þ Kuangetal.,2000a
BPSc(ISS) active þ þ Morrow,pers.comm.
Epidendrum Malachite(Salyut6) open (cid:1)d (cid:1) Nechitailo&Mashinsky,1993
Seeds
Arabidopsis Svetoblok(Salyut6) closed þ (cid:1) Kordyumetal.,1983
Phyton(Salyut7) passive þ þ MerkysandLaurinavicius,1983
AdvancedAstrocultured active þ þ Stankovicetal.,2001
Pisum Oasis(Salyut6) open (cid:1) (cid:1) NechitailoandMashinsky,1993
Brassica Svet(Mir) openw/fan þ þ Musgraveetal.,2000
Triticum SvetoblokM(Mir) passive þ (cid:1) Mashinskyetal.,1994
Svet(Mir) openw/fan þ (cid:1) Stricklandetal.,1997
Svet(Mir) openw/fan þ þ Binghametal.,1999
aThehardwareinthiscasewasanopenbeakercontainingmoistsoil.
bPlantGrowthFacility.
cBiomassProductionSystem.
dCommercialhardwareusedontheInternationalSpaceStation.
3
4
Fig.1. Evolutionin planthardwaredesignhas madeplantreproductionduringspaceflightareliable
occurrence.(A)ThefirstsophisticatedhardwareusedtogrowArabidopsisinspacewasSvetoblok(on
Salyut6),asmall,hermeticallysealedplantgrowthchamberthatcouldbeclippedontoalightinthe
cabin.ThemetalcylinderwouldsnapoverthechamberforprotectionduringtransportbacktoEarth.
Lowlightandlackofgasflowwithinthechamberwereprobablyresponsibleforthereproductivefailure
reported (Kordyum et al., 1983), as absence of convective air movement in microgravity makes such
unventilatedchambersproblematicforlong-durationgrowthofplants.(B)MatureArabidopsisplants
were grown in the Advanced AstrocultureTM unit on the ISS in 2001. The entire root tray is visible.
Photo courtesy of Bratislav Stankovic, Wisconsin Center for Space Automation and Robotics
(WCSAR).(C)BrassicaandwheatweregrownintheBiomassProductionSystemontheISSin2002.
The BPS is the precursor for the Plant Research Unit, a habitat designed for use in the planned
Centrifuge Accommodation Module of ISS. (D) Images acquired during flight were relayed to the
groundinrealtime,makingdailymonitoringofreproductionbyBrassicaintheBPSpossible.PhotosC
andDcourtesyofRobertMorrow,OrbitalTechnologies,Inc.,Madison,WI.
The first successful plant life cycle in microgravity occurred on Salyut 7 in a
small, ventilated plant growth chamber called Phyton. Arabidopsis seeds sown
in microgravity on an agar surface by a ‘‘seed shooter’’ grew into plants that
eventuallyfloweredandproducedseedsthemselves(MerkysandLaurinavicius,
1983).
Attempts to design hardware to support more vigorous plant growth
resulted in a variety of units. Bulgarian-made Svet provided a well-ventilated
environment with a fan that drew cabin air through the unit. Subsequent
upgrades resulted in brighter lights, a root module with regulated water
injection, and a gas exchange module that permitted measurement of
rootzone moisture and canopy carbon dioxide gas exchange. Using Svet,
5
Bingham et al. (1996) showed that a persistent problem in plant hardware
designisthetendencytooverwaterinmicrogravity,sinceadoseofwateronthe
ground resides lower in the root matrix than it does in microgravity. Using
moisture sensors that reported actual water status in the root matrix volume,
they were able to grow plants full term in Svet. Wheat (Bingham et al., 1999)
and Brassica (Musgrave et al., 2000) would be grown from seed to maturity in
this unit prior to the deorbiting of the Mir space station in 2000.
Units with more modestly sized head spaces were developed for use in
the shuttle mid-deck. Of these, the Plant Growth Unit and Plant Growth
Facility both supported studies on reproductive development in Arabidopsis
(Kuang et al., 1995, 1996a,b) and Brassica (Kuang et al., 2000a), respectively.
Individual small chambers (5 or 6) within the units each supported six pre-
grown plants through flowering and early reproductive development over
periods of up to 16 days on orbit.
Two plant growth hardware units originally designed for the shuttle are
currently in use for long-duration plant growth studies on the International
Space Station. The Advanced Astroculture Unit (Fig. 1b) grew Arabidopsis
throughtoseedsetonISSin2001(Stankovicetal.,2001).In2002theBiomass
Production System (Fig. 1c) supported growth by the larger plant, Brassica
(Fig. 1d), through seed production. The BPS is the precursor design for the
Plant Research Unit hardware that will be housed in the Centrifuge
Accommodation Module on ISS at assembly complete (Morrow et al., 2001).
Transition from vegetative to flowering stage
As chronicled in the previous section, researchers interested in plant
reproductive development in microgravity were initially unable to confirm
any specific role for gravity in plant reproduction because of general problems
inthegrowthofplantsinspace.Theplantgrowthoverextendedperiodsthatis
necessary for reproduction to occur was not possible, and plants frequently
diedinthetransitionfromvegetativetoreproductivestage(Table1).Evenwith
experimentswiththesmallplantArabidopsis,whichhasamongthelowestlight
and nutrient requirements of any higher plant, delays in transition to the
reproductive stage were the norm in microgravity.
Weconductedaseriesofexperimentstodetermineiftheproblemwithplant
reproduction in space was just a consequence of overall poor plant growth
in microgravity, or if there was a specific effect of microgravity on the
reproductiveapparatusperse.UsingArabidopsisplantsthathadbeengrownto
the pre-flowering stage, we quantified the development of flowers during three
experiments in microgravity: 6 (Kuang et al., 1995), 10 (Kuang et al., 1996a)
and 11 (Kuang et al., 1996b) days in duration on the space shuttle.
Table 2 summarizes details of the three experiments, and the floral production
in each on a per-plant basis. Unlike previous experiments in which flowering
was delayed, these experiments, using plants at the pre-flowering stage at the
6
Table2
FlowerproductioninmicrogravitybyArabidopsisplantslaunchedatthepre-floweringstage,compared
withcorrespondinggroundcontrols.Resultsofthreeexperiments,ofdifferentdurations,inthePlant
GrowthUnit
Flowersperplant
Flight# Duration(days) Flight Ground Configuration
STS-54 6 16.1 16.1 Closedchambers
STS-51 10 44.1 39.8 ClosedþCO
2
STS-68 11 79.5 77.5 WithAESa
aAirExchangeSystem,providedaslowexchange(90ml/min)ofchamberairwithfilteredairfromthe
crewcabin.
time of introduction to the microgravity environment, yielded flower
production at the same magnitude and schedule as in the ground control
(Table 2 and Fig. 2).
Despite the equivalent rates of floral initiation in these three experiments,
success of the transition to reproductive development was highly dependent
uponotherfactorslistedinTable2andFigure3.IntheexperimentonSTS-54,
bothandroeciumandgynoeciumabortedatanearly stage(Kuangetal.,1995;
andseepertinentsectionsbelowfordetails).Analysisoffoliarmaterialshowed
a significantly lower amount of carbohydrate in the spaceflight material
(Musgraveet al., 1998). In the next experiment on STS-51, supplementation of
the closed headspace of the Plant Growth Chamber with high carbon dioxide
(8000 ppm) raised the foliar carbohydrates, and early development of both
androecium and gynoecium proceeded normally, however without pollination
due to high humidity in the closed chamber (Kuang et al., 1996a). In the third
experimentonSTS-68,aflowthroughventilation systemwas usedtomaintain
a supply of carbon dioxide to the foliage, resulting in higher concentrations of
foliar starch. Again, androecium and gynoecium development proceeded
normally, and the flow through system reduced humidity sufficiently to allow
anther dehiscence, pollination, and subsequent embryo development (Kuang
et al., 1996b).
The important role played by the chamber environment in promoting
successful transition from vegetative to reproductive development is illustrated
in Figure 3. Note that the closed chambers in both the experiments on STS-54
and STS-51 resulted in significantly lower soluble carbohydrates in the
spaceflightmaterial,whiletheflow-throughsysteminuseduringtheexperiment
on STS-68 allowed comparable amounts of soluble carbohydrates to
accumulate in the foliage in flight and ground control. Starch concentration
inthefoliagefollowedasimilarpattern.Althoughtheclosedsystemssupported
reproductive development in 1-g, lack of convective mixing in microgravity
significantly slows the resupply of metabolic gases to the plant.
7
Fig.2. FloraldevelopmenttimingwasthesameinspaceflightandgroundcontrolArabidopsisplantsas
judgedbythepost-flightcensusofbudsizesinexperimentsonSTS-54(A)andSTS-51(B),respectively.
Approximatelytwiceasmanybudswerepresentafterthe10-dayexposuretomicrogravity(onSTS-51)
asfollowingthe6-dayexposure(onSTS-54).Plantswereatthepre-floweringstageatthetimeoflaunch.
Table 1 highlights the important role played by ventilation in fostering
successful plant reproduction in microgravity (Musgrave et al., 1997). Clearly,
hardware providing an open or flow-through system is needed in order to
permit successful transition from the vegetative to reproductive stage and
subsequentseeddevelopment.Asdescribedinthissection,carbohydratestatus
of the plant in transition from the vegetative to reproductive stage must be
sufficient to support the energy demands. This is readily accomplished with an
open system if the watering demands can be met but has been problematic in
closed chambers in microgravity. Recently, Porterfield et al. (2000) and Kitaya
et al. (2000) demonstrated the important role played by gravity in maintaining
the small-scale gas transport necessary to sustain metabolic processes.
8
Fig.3. AseriesofthreeexperimentswithArabidopsisinthePlantGrowthUnitonSTS-54,STS-51and
STS-68 established a link between carbohydrate status of the foliage and successful transition to the
reproductivestageinthespaceflightenvironment.RefertoTable2andthetextforadditionaldetails.
BasedondatafromMusgraveetal.(1998).
Anther development and pollen quality
Intheseriesofexperimentsdescribedabove,manyassayswereemployedpost-
flight to assess the success of every aspect of the reproductive process. Anther
development aborted at an early stage in Arabidopsis during 6 days of
spaceflightexposureonSTS-54(Fig.4),providingverysimilarmaterialtothat
