On earth, gravity affects the distribution of the plant growth hormone, indole-3-acetic acid (IAA), in a manner such that the plant grows into a normal vertical orientation (shoots up, roots down). How the plant controls the amount and distribution of IAA is only partially understood and is currently under investigation in this laboratory. The question to be answered in the flight experiment concerns the effect of gravity on the concentration, turn over, and distribution of the growth hormone. The answer to this question will aid in understanding the mechanism by which plants control the amount and distribution of growth hormone. Such knowledge of a plant's hormonal metabolism may aid in the growth of plants in space and will lead to agronomic advances

Bandurski, Robert S.

Desrosiers, Mark

Jensen, Philip

Schulze, Aga

English

NASA Technical Reports Server

N90-13947
EFFECTS OF MICROGRAV1TY ON GROWTH HORMONE CONCENTRATION AND
DISTRIBUTION IN PLANTS
Robert S. Bandurski,
Aga Schulze, Philip Jensen and Mark Desrosiers
Departments of Botany & Plant Pathology
Michigan State University
East Lansing, Michigan 48824-1312
ABSTRACT
On earth, gravity affects the distribution of the plant growth hormone,
indole-3-acetic acid (IAA), in a manner such that the plant grows into a
normal vertical orientation -- shoots up, roots down. How the plant controls
the amount and distribution of IAA is only partially understood and is
currently under investigation in this laboratory. The question to be answered
in the flight experiment is: "How does gravity affect the concentration, turn
over, and distribution of the growth hormone?" The answer to this question
will aid in understanding the mechanism by which plants control the amount
and distribution of growth hormone. Such knowledge of a plant's hormonal
metabolism may aid in the growth of plants in space and will lead to agronomic
advances.
INTRODUCTION
Ground-based studies:
The shoot of a young plant, placed in a horizontal position, grows back to
a vertical orientation (Figure 1). The response begins within minutes after
the plant is placed horizontally and vertical orientation is restored at a rate of
1 o x min -1 (Bandurski et al, 1984). How the plant perceives gravity and how
the gravity signal is transduced into an asymmetric growth response is only
partially understood (Wilkins, 1984; Bandurski et al, 1986a). The plant's
gravity response, and the lack of that response in micro-gravity, will be
important in attempts to grow plants under micro-gravity conditions.
Gravity detection:
Owing to the pervasiveness of gravity, it is likely that plants sense
gravity by more than one mechanism. For example, some plants may utilize
the settling of dense starch grains, statoliths, to the bottom of the cell as a
gravity-sensing mechanism (Bandurski et al, 1984; Sievers & Hensel, 1982).
However, there is also evidence that a mutant plant, lacking
phosphoglucomutase in its chloroplasts -- and thus lacking starch-filled
statoliths -- can sense gravity almost as readily as normal plants (Caspar T,
Sommerville C. 1988. Personal Communication).
This mutant is detecting gravity without dense starch grains. Statoliths
may perceive gravity in some plants but they are obviously not the only
mechanism for gravity perception. For example, a mechanism for gravity
sensing, not involving the settling of dense particles, has been proposed
(Bandurski et al, 1986a). In this mechanism, any distortion of the cells' shape
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or of the microtubular structures in the cytoplasm of the cell could be used
for gravity sensing.
Membrane depolarization:
Despite the uncertainties regarding gravity sensing, it is known that
both gravity and light stimuli result in membrane depolarization. This
phenomenon has been studied for more than 50 years (Wilkins, 1984; Dolk,
1933) and has recently been studied elegantly by Tanada (1983) and by Sievers
and colleagues (1Bandurski et al. 1988. In Press). Membrane depolarization
is the first detectable response of a plant to a gravitational stimulus,
occurring within 8 sec after the stimulus is given (1Bandurski et al., 1988. In
Press). It is the rapidity of the depolarization response and its induction by
two such diverse stimuli as gravity and light which suggests that membrane
depolarization is an integral part of the tropic response.
Hormone asymmetry:
The next detectable response following membrane depolarization is an
asymmetric distribution of the plant growth hormone, indole-3-acetic acid
(IAA) (Bandurski et al, 1984; Bandurski et al, 1986a; l Bandurski et al, 1988).
The central focus of our research has been the question, "How does the plant
transduce a membrane depolarization into an asymmetric distribution of
IAA?" We believe that emphasis on the chemical asymmetry, rather than on
the more complex issue of growth asymmetry, will facilitate attaining an
understanding of the gravity response at a molecular level.
A wgrking theory:
This laboratory has developed a working theory for the transduction of
the gravity stimulus into an asymmetric distribution of IAA. We postulate that
a change in the orientation of the plant with respect to the gravitational field
induces a membrane depolarization as discussed above (Bandurski et al, 1986a;
Tanada, 1983; Behrens et al, 1985; 1Bandurski et al, 1988) Next, we postulate
that membrane depolarization open and/or closes plasmodesmatal channels
between the plants vascular tissue and the surrounding cortical and
epidermal tissues. IAA, calcium, and other substances, can then flow
selectively into the bottom side of a horizontal stem inducing a more rapid
growth rate on the bottom side of the stem. The plant would then grow into its
normal vertical orientation. Evidence for this theory is reviewed in
references Bandurski et al (1986a) and 1Bandurski et al (1988).
Growth asymmetry:
Growth is complex involving the regulated occurrence of perhaps
thousands of reactions. However, in our experimental system, employing 5
day old seedlings of corn (Zea mays), growth is an arithmetic function of IAA
1Bandurski RS, Schulze A, Desrosiers M, Jensen P, Epel B, and Reinecke D.
1989. Relationship between stimuli, IAA, and growth. In: Plant Growth
Substances. 1988. Pharis R, Rood R, Eds. In press
122
concentration. Thus, we confirm and extend the earlier concepts (Went &
Thimann, 1937) that growth is controlled by IAA and that an IAA asymmetry
will result in a growth asymmetry.
Summary of ground-based research:
The intent of this laboratory has been to attempt to link membrane
depolarization to a chemical asymmetry within the plant. The chemical
asymmetry could then result in a growth asymmetry such that the plant
grows back into its normal orientation.
In summary, the sequence of events is believed to be: 1) sensing of the
gravitational stimulus; 2) transduction of the stimulus into a membrane
depolarization; 3) transduction of the membrane depolarization into a
chemical asymmetry; and 4) transduction of the chemical asymmetry into
asymmetric growth.
Flight program:
We do not have a theoretical basis for predicting the effect of
microgravity on the growth hormone IAA other than our working theory.
We know that at 1 g, IAA becomes asymmetrically distributed within a
horizontally-placed plant. We believe this asymmetric distribution to be
owing to selective movement of IAA from the vascular stele into the
surrounding cortical tissues with more IAA coming from the lower side of the
stele. The flight experiment will tell us whether the channels between stele
and cortex are open or closed in the absence of the gravitational stimulus.
This knowledge will be of value in understanding how plants regulate their
endogenous IAA levels and may help in the growing of the plants in space.
RESULTS
Synopsis of the experimental protocol:
The plant seeds (kernels) are wrapped in filter paper, loaded into
canisters and water added 12 h prior to launch. Two canisters and one LN2
freezer are placed in each of two middeck lockers. The plants are allowed to
grow for 108 h (total hydration plus growth time equals 120 h) at which time
two of the canisters are permitted to grow until shuttle landing. Upon
landing the two unfrozen canisters and the two prefrozen canisters are put
into a 35 VHC, Taylor-Warton liquid nitrogen refrigerator. After several hrs,
the frozen canisters are transferred to a dry-ice shipping container, loaded
with solid CO2 and sent to East Lansing for analytical studies. In East Lansing,
the plants will be dissected into roots, seed, and shoot tissue and ground in
aqueous acetone for extraction and determination of free and ester IAA.
Experimental design:
The plants must be grown in darkness, in microgravity, and frozen prior
to landing. We have designed the plant growth container to minimize crew
handling time and eliminate the possibility of plant material or moisture
escaping into the mid-deck of the shuttle. Figure 6 shows a photograph of the
canisters used for plant growth. There are two compartments to each
123
canister. Table I summarizesthe weight, contents and dimensionsof the
canisters.
TABLE I
Canister length 335.0
Canister diameter 82.0
Canister weight 860.0
(There are 2 compartments per canister)
14 Teflon sleeves (7.07g ea) X2=
28 filter papers (2.15g ea) X2=
(Two filter papers per kernel)
14 kernels(0.197g ea) X2=
water(8 m/kernel) X2=
Total per canister
mm
mm
g
19.08 g
121.0 g
5.52 g
224.0 g
1408.52 g
One fully charged LN2 14,870.0 g
So 4 canisters would weigh 5634 g and 2 fully charged LN2's would weight
29740g for a total experiment weight of 35374 grams.
t3a_ exchange:
The canisters are vented through 4 light baffled holes to permit gas
exchange with the air of the middeck locker. As can be seen in Figure 2, the
venting is adequate to prevent the build-up of CO2 and there is no benefit by
adding an ethylene absorbent. Figure 3 shows that the venting is adequate to
prevent depletion of oxygen and again there is no benefit by adding an
ethylene absorbent.
We conclude that the canisters are adequately vented for the growth of 28
seedlings during 120 h growth period.
The assay for IAA:
An important part of both the ground-based studies and the flight
program has been the development of a senstive and reliable assay procedure
for IAA. Owing to the lability of IAA, its presence in low (10 -8 M)
concentration, and the presence of 10 -3 M interfering phenyipropene acids,
an internal standard must be employed. Colorimetric, fluorometric, and
radioimmunoassays have proven useless (Pengelly & Bandurski, 1983; Cohen
et al, 1987). The following assay has proven to be sensitive and accurate and
provides proof that it is really IAA that is being measured. We originally
synthesized 4,5,6,7-tetra deutero IAA as an internal standard (Magnus et al,
1980) but this has now been replaced by IAA labeled with 6 atoms of 13C in the
benzene ring portion of the indole nucleus (Cohen et al, 1986).
Extraction of IAA from the plant tissue:
The plants from the two canisters frozen in space will be separated into
shoots, seeds, and roots, weights recorded and the plants then homogenized in
sufficient acetone to make the final acetone concentration 70%. (All
percentages are vol/vol.) The plants from the remaining two canisters will
124
be treatedsimilarly and used as "controls" since they will have had, at least, 90
minutes of recovery time at one, or more, g. Ground controls will be similarly
treated. The homogenateswill be filtered, residueswashedand weighed, and
the volume of the aqueousacetoneextracts determined. Two thirds of each
extract will be used for the determinationof free IAA and one third will be
used for determinationof esterified IAA. To each extract a known amount of
13C IAA will be addedin amountssuch that the 13C IAA will rangebetween1
to 10 times the plant IAA. In addition,about540,000DPM of 22.6 Ci/mmole
tritiated IAA will be added. This amount(1,884picogramsof 5- 3H-IAA is one
mass unit heavier then the plant's IAA and further is only 9.4% of the IAA of
a 1 g samplecontaining20 ng of IAA per g) and so does not interfere with the
assaybut facilitates locating peaks on chromatograms. The aqueousextracts
are concentratedin vacuo, made to 50% aqueous ethanol, applied to a 2 ml bed
volume DEAE-acetate column and the volumn washed with 10 column volumes
of 50% ethanol-water to remove non-anionic compounds. The column is then
gradient eluted with 50 ml of 50% acetic acid in the mixing flask and 50 ml of
50% aqueous ethanol containing 5% acetic acid in the reservoir. IAA elutes at
about 20 ml. The samples for determination of free plus ester IAA will have
been treated similarly except that the samples will first be hydrolyzed with 1
M NaOH for 15 min at 22°C, then adjusted to pH 2.5, and the IAA extracted into
ether, concentrated, taken up in 50% aqueous ethanol and treated as above.
The pooled IAA containing sample is reduced to near dryness (50 I.tl of
capryl alcohol was added to prevent foaming and to prevent the sample from
going to dryness) in vacuo, taken up in 200 _tl of 50% aqueous ethanol and
applied to a 4.8 mm X 250 cm C18 reverse phase HPLC column. Development is
with 30% aqueous ethanol containing 0.1% acetic acid. The radioactive
sample is collected at about 12 ml, dried in vacuo, taken up in 100 txl of
methanol, methylated with 300 txl of ethereal diazomethane (Bandurski et al,
1986b), dried and taken up in 20 to 50 _tl of acetonitrile for GC-MS.
Gas chromatography is on a 12.5 m 0.2 mm wall coated OV-17 column butt
connected to 15 cm of 0.5 mm uncoated quartz pre-column and using direct on
column injection. The GC-MS is the Hewlett-Packard 5890-5970 table top
model. As shown in Figure 4, the chromatography is very good, and as shown
in Figure 5, the ratio of amounts of material at masses 189 and 195 and 130 and
136 is easily determined. Mass 189 is the molecular ion of methyl IAA and 136
is the quinolinium ion of 6C 13 IAA. The ratios of ions at 195/189 and 136/130
agree within 0.1% giving assurance that only pure IAA is being measured.
EXPECTED BENEFIT_
As indicated above, there is no adequate theoretical basis for predicting
the effects of gravity, or the lack of gravity, on biological systems.
Mammalian systems, although of primary importance in terms of humans in
space, appear terribly complicated and may be less suited than plants and
microorganisms for attaining an understanding of gravity effects at the
molecular level.
We believe that our system, utilizing 5 day old dark-grown corn plants is
possibly the best eucaryotic plant system available. It is a closed system since,
in darkness, the plants must obtain all of their nutrients and their growth
hormone, IAA, from seed (Bandurski et al, 1986a; 1Bandurski et al, 1988;
Bandruski et al, 1986b; Reinecke & Bandurski, 1987). Further, we have
evidence that the targets for the gravitational response on earth, are the
125
plasmodesmatalchannels connecting the vascular tissues of the stele with the
cortical and epidermal tissues.
We have not completedour electrophysiologicalstudies and so we can not
predict whether the plasmodesmatalchannels will be open or closed in
microgravity. However, following the flight experimentwe will be able to
measurethe size of the plants, their dry weight, how much IAA and IAA
conjugatesare in the shoot and, importantly, the amountof IAA and IAA
conjugates left in the shoot. Such knowledge will provide another important
set of facts which must be fit into any working theory for the molecular basis
of the gravity effect on plants.
Such knowledge will be of practical value to terrestrial agriculture.
Whether this knowledge will result in important advancesin space
technology is unknown. If there are no important microgravity effects, it
will be of aid to the space station program to know there are no fundamental
hormonal problems that prevent a successful agriculture in space. If there
are microgravity effects than it is possible that a technology based upon
substitution of electrical potentials for the gravitational stimulus might be of
practical value in facilitating a space based agriculture (Desrosiers &
Bandruski, 1988). Either result must ultimately fit into theories concerning
how a plant regulates it endogenoushormone levels.
OBJECTIVE
We know that hormones control growth and development, but what
controls the amount of the hormone? That is the ultimate objective of this
experiment.
REFEREN_-'w_
Bandurski, R.S., A. Schulze, P. Dayanandan, and P.B. Kaufman. 1984. Plant
Physiology. 74:284.
Bandurski, R.S., and A. Schulze, W. Domagaiski. 1986a. XXVI COSPAR Adv Space
Res. 6:47.
Bandurski, R.S., A. Schulze and D.M. Reinecke. 1986b. Biosynthetic and
metabolic aspects of auxins, p83. In: Plant Growth Substances. 1985.
M, Ed. Springer-Verlag, Heidelberg.
Bopp
Behrens, H.M., D. Gradman, and A. Sievers. 1985. Planta. 163:463.
Cohen, J.D., B.G. Baldi and J.P. Slovin. 1986. Plant Physiology. 80:14.
Cohen, J.D., M.G. Bausher, K. Bialek, J.G. Buta , G.F.W. Gocal, L.M. Janzen, R.P.
Pharis , A.N. Reedand and J.P. Slovin. 1987. Plant Physiology. 80:961.
Desrosiers, M.R. and R.S. Bandurski. 1988. Plant Physiology. 87:874.
Dolk, H. 1933. Rec Trav Bot Neerl. 33:509.
Pengelly, W.L. and R.S. Bandurski. 1983. Plant Physiology. 73:445.
Magnus, V., A. Schulze, and R.S. Bandruski. 1980. Plant Physiology. 90:961.
126
Reinecke D.M.and R.S. Bandurski. 1987. Auxin biosynthesisand metabolism.
In: Plant hormonesand their role in plant growth and development.
Davies P, Ed. Nijhoff-Junk Publishers,Netherlands. p24.
SieversA. and W. Hensel. 1982. The natureof graviperception, pp 497-506. In:
Plant Growth Substances. WareingPF, Ed. AcademicPress,New York.
Tanada,T. 1983. Plant, Cell, Environ. 6:69.
Went, F.W. and K.V. Thimann. 1937. Phytohormones. MacMillan New York.
Wilkins, M.B. 1984. Gravitropism. pp. 153-185. In: Advanced Plant
Physiology. Wilkins MB, Ed. Pitman London.
127
Fig. 1. Time lapse photograph of a seedling of Z. mays during gravitropic
curvature. The intial photograph was taken just as the seedling was
placed horizontally. Successive photographs are taken at 15-min
intervals. The India-ink marked 'N' indicates the node between the
coleoptile and mesocotyl..
ORIGINAL PAGE
BLACK AND WHITE PHOTOGRAPH
128
5&
o
u 4
SEALED
SEALED + SODA SORB
VENTED + SODA SORB
0 24 48 72 96 120 144
TIME, hr
Fig. 2. Venting the canister prevents the build up of carbon dioxide so that a
CO2 absorbent such as soda sorb need not. be added. Each compartment of
the canister contained 14 germinating kernels of corn (Zea mays) for
120 h.
129
22
20
18
16
14
&12
0
10
v
O SEALED
A SEALED + ADS
• VENTED
[] VENTED + ADS
I I I I I I
24 48 72 96 120 144
TIME, hr
Fig. 3. Venting the canister prevents oxygen depletion. Addition of an
ethylene and carbon dioxide absorbent did not change the per cent
oxygen in the gas phase. Each compartment of the canister contained
14 germinating kernels of corn (Zea mays) for 120 h.
4 10 5
TIC OF DATA:ASCCRTE1.D
uJ
tO
z
<
£3
Z
Pt_
<
,,, i ..... L .... ,:, .-.--__
3 4 5 6 7
TIME, min
Fig. 4. Total ion current monitored as a function of retention time on a 12.5m
OV-17 WCOT. As can be seen the purified and methylated IAA from the
plant is almost free of any contaminants. This, possibly excessive,
purification prior to GC/MS assay keep the injector and columns clean
and improves our day to day sensitivity.
130
BLACK A_D _'"_111;L Pi_O]OGRAPH2.4/105
2.2 1_36
2.0 /I1
1.8
_ 1.6
_ 1.4
_ 1.2
< 1.0
30
8:×104 I_
6.00 6.10 6.20 6.30
TIME, min
Fig. 5. Monitoring of massess 195 and 189, the molecular ions of the methyl
ester of 6C13 IAA and plant IAA, and 136 and 130, the quinolinium ions
of 6C13 IAA and plant IAA. Agreement of the ratio 195/189 and 136/130
is usually within 0.1% giving assurance that the compound being
measured is, in fact, IAA.
Fig. 6. A photograph of the plant canister separated into its two compartments.
The lid screws into the top of one compartment which then screws into
the bottom compartment to comprise the canister of two compartments.
Construction is of anodized aluminum.
131



Effects of microgravity on growth hormone concentration and distribution in plants

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