Chapter 15
Lignification in Young Plant Seedlings Grown on Earth and Aboard the Space Shuttle Joe R. Cowles, R. LeMay, G. Jahns, W. H. Scheld, and C. Peterson Department of Biology, University of Houston, Houston, TX 77204-5513
The space shuttle era has provided an opportunity for investigators to conduct experiments in a microgravity environment. Two shuttleflights,STS-3 and STS-51F, each contained an experiment designed principally to determine whether young plant seedlings exposed to microgravity had reduced lignin content in comparison to seedlings grown at one gravity. Three different plant species, pine, oats, and mung beans, were exposed for eight days to the microgravity environment of the shuttle. The lignin content of in-flight seedlings was less than the control seedlings in all seven sets of seedlings included in these two experiments. In five sets of seedlings, the reduction in lignin content inflightseedlings ranged from 6 to 24% and was statistically significant. In addition, the activity of two enzymes involved in lignin synthesis, phenylalanine ammonia lyase and peroxidase, were significantly reduced in pine seedlings. It was therefore concluded that microgravity, as perceived by young plant seedlings, results in reduced lignin synthesis. T h e stems of most higher p l a n t s grow u p w a r d i n a n a n t i g r a v i t a t i o n a l d i r e c t i o n , a n d are s u p p o r t e d b y the s t r u c t u r a l p o l y m e r s synthesized a n d deposited i n the cell walls. T h e p r i n c i p a l s t r u c t u r a l components o f p l a n t cell walls are l i g n i n , cellulose a n d hemicelluloses. T h e biosynthesis o f at least one o f these p o l y m e r s , l i g n i n , is believed t o be effected b y g r a v i t y (1-4). T h e evidence t h a t g r a v i t y is a n i m p o r t a n t factor i n l i g n i n synthesis has a c c u m u l a t e d f r o m a n u m b e r o f different e x p e r i m e n t s over several years (25). U n t i l recently, however, investigators were u n a b l e t o d e t e r m i n e d i r e c t l y whether g r a v i t y was required for l i g n i f i c a t i o n because a zero o r m i c r o g r a v i t y was n o t available for a n extended p e r i o d o f t i m e . T h i s has changed 0097-6156/89/0399-0203$06.00/0 © 1989 American Chemical Society
204
PLANT C E L L W A L L P O L Y M E R S
w i t h the advent of the space s h u t t l e where scientific e x p e r i m e n t s c a n be exposed to at least the m i c r o g r a v i t y e n v i r o n m e n t of the space s h u t t l e . W e were p r o v i d e d a n o p p o r t u n i t y to fly two p l a n t e x p e r i m e n t s o n the s h u t t l e . T h e first e x p e r i m e n t was o n Space T r a n s p o r t S y s t e m - 3 ( S T S - 3 ) , w h i c h was l a u n c h e d o n M a r c h 2 1 , 1982. T h e second e x p e r i m e n t was p a r t of S p a c e l a b II, w h i c h was flown a b o a r d S T S - 5 1 F t h a t was l a u n c h e d o n J u l y 29, 1985. B o t h of these flights were for eight days d u r a t i o n . T h e p r i n c i p a l e x p e r i m e n t a l o b j e c t i v e was t o e s t a b l i s h the effect of m i c r o g r a v i t y o n l i g n i f i c a t i o n i n higher p l a n t seedlings, a n d was p a r t o f a n overall g o a l to u n d e r s t a n d the effect o f g r a v i t y o n l i g n i f i c a t i o n i n higher p l a n t s . T h i s paper r e p o r t s a s u m m a r y of l i g n i f i c a t i o n values a n d p h e n y l a l a n i n e a m m o n i a lyase ( P A L ) a n d peroxidase a c t i v i t y f r o m the S T S - 3 a n d S T S - 5 1 F e x p e r i m e n t s . T h e results show a r e d u c t i o n i n b o t h l i g n i f i c a t i o n a n d e n z y m e a c t i v i t y i n flight seedlings as c o m p a r e d to one g r a v i t y c o n t r o l seedlings. Materials, Procedures and Methods Flight Hardware. T h e e x p e r i m e n t s were contained i n m i n i g r o w t h u n i t s referred to as P l a n t G r o w t h U n i t s ( P G U ' s ) . B a s i c a l l y the P G U ' s consisted of a c a v i t y for g r o w i n g p l a n t s , a light source, a passive t e m p e r a t u r e c o n t r o l s y s t e m a n d a n a u t o m a t e d r e c o r d i n g s y s t e m (6) ( F i g . 1). T h e seedlings were g r o w n i n s m a l l p l a n t g r o w t h chambers ( P G C ' s ) t h a t fitted w i t h i n the c a v i t y o f the P G U ' s ( F i g . 2). E a c h P G U a c c o m m o d a t e d u p to 6 P G C ' s . T w o flight-qualified P G U ' s were b u i l t b y L o c k h e e d M i s s i l e s a n d Space C o . a n d N A S A A m e s Research C e n t e r , respectively. In the S T S - 3 e x p e r i m e n t , one P G U was flown a n d the other was used to grow c o n t r o l seedlings i n real t i m e at N A S A K e n n e d y Space C e n t e r ( K S C ) . In the S T S - 5 1 F e x p e r i m e n t b o t h P G U ' s were flown a n d subsequently used for the post-flight c o n t r o l e x p e r i m e n t at N A S A K S C . Pre-flight Activities. T h e pre-flight a c t i v i t i e s at N A S A K S C were i n i t i a t e d 10 a n d 15 days ahead of the scheduled S T S - 3 a n d S T S - 5 1 F launches, respectively. I n b o t h e x p e r i m e n t s m u n g b e a n a n d oat seeds were p l a n t e d w i t h i n 16 hours of scheduled l a u n c h . In a d d i t i o n to p l a n t e d seeds, the exp e r i m e n t s i n c l u d e d 4 - d a y - o l d pine (Pinus elliotti E n g e l m ) seedlings i n the S T S - 3 e x p e r i m e n t a n d 4- a n d 10-day-old pine seedlings i n the S T S - 5 1 F exp e r i m e n t . In the S T S - 3 e x p e r i m e n t 2 P G C ' s were devoted to each p l a n t species (16 seeds or seedlings each). I n the S T S - 5 1 F e x p e r i m e n t 3 P G C ' s were c o m m i t t e d to each age g r o u p of pine seedlings a n d 2 a n d 4 P G C ' s were devoted to oats (Avena sativa L . cv. G a r r y ) a n d m u n g beans ( Vigna radiata), respectively. P r e - l a u n c h a c t i v i t i e s i n c l u d e d p l a n t i n g m u n g b e a n a n d oat seeds, assembling P G C ' s , e x c h a n g i n g a t m o s p h e r i c gas i n the sealed P G C ' s w i t h a defined gas ( 2 1 % 0 , 300 p p m C 0 a n d balance N ) , P G C p h o t o g r a p h y , a n d l o a d i n g P G C ' s i n t o P G U . T h e loaded P G U ' s were t r a n s p o r t e d to the l a u n c h p a d a n d placed i n t o the s h u t t l e m i d - d e c k . 2
2
2
Flight Activities. T h e P G U ' s were designed to require m i n i m a l in-flight a t t e n d a n c e . S h u t t l e crew t i m e , however, was requested to read a n d record P G C t e m p e r a t u r e s a n d to check flight e q u i p m e n t 2 to 3 times daily. O n the S T S - 3 m i s s i o n , the t e m p e r a t u r e d a t a were r o u t i n e l y voiced d o w n a n d used
Plant growth chamber base 6 places
Thermistor 6 places
Sample/purge p o r t - 2 ploces/chamber
Air inlet
Door
Master power switch/indicator light
PGU power input
Heater Data output (optional)
Access door
Captive fasteners
F i g u r e 1. A d i a g r a m m a t i c s k e t c h of the P G U .
Temperature display (°C) Chamber temperature select Temperature display enable Chamber temperature warning indicator Warning disable Lamp status Lamp mode select Recorder malfunction
Plant growth chamber cover - 6 places
ILC lamp assy
Tape recorder
package
- Electronics
Battery
206
PLANT C E L L W A L L POLYMERS
Base ο Ο Ο
F i g u r e 2. A d i a g r a m m a t i c sketch of the P G C .
15.
COWLESETAL.
Seedlings Grown on Earth & on Space Shuttle
207
i n c o n d u c t i n g the c o n t r o l e x p e r i m e n t . I n a d d i t i o n to r e a d i n g t e m p e r a t u r e s a n d c h e c k i n g h a r d w a r e , the S T S - 5 1 F flight crew also took p h o t o g r a p h s of the p l a n t seedlings a n d s a m p l e d a t m o s p h e r i c a i r i n the P G C ' s . Post-Flight Activities. T h e S T S - 3 flight l a n d e d at a n a l t e r n a t e l a n d i n g site near W h i t e S a n d s , N e w M e x i c o , where a t e m p o r a r y l a b o r a t o r y was established i n a m o t o r h o m e . T h e P G U a r r i v e d to the l a b o r a t o r y a b o u t 75 m i n p o s t - l a n d i n g , where observations, P G C a i r s a m p l i n g a n d a n a l y s i s , p h o t o g r a p h y , a n d p l a n t m e a s u r i n g a n d p a c k a g i n g was c o n d u c t e d . A b o u t 4 hr after l a n d i n g the seedlings were packed i n i n s u l a t e d containers s u r r o u n d e d b y c o l d packs for r e t u r n to the P I l a b o r a t o r y for a n a l y s i s . U p o n a r r i v a l ( a b o u t 13 h r p o s t - l a n d i n g ) , seedlings selected for l i g n i n a n d e n z y m e a n a l ysis were f u r t h e r processed. T h e s t e m sections used for e n z y m e a n a l y s i s were frozen for l a t e r a n a l y s i s . A l l stems were measured a n d cut i n t o sect i o n s . T h e s t e m sections used for l i g n i n a n a l y s i s were t r e a t e d w i t h 1.5 m l a c e t o n e / 1 % H C 1 to b e g i n a 24 h r d i g e s t i o n . T h i s phase o f processing was c o m p l e t e d about 18 h r p o s t - l a n d i n g . T h e S T S - 5 1 F flight l a n d e d at N A S A D r y den, E d w a r d s A i r Force B a s e , C a l i f o r n i a , where a t e m p o r a r y l a b o r a t o r y h a d been e s t a b l i s h e d . T h e a m o u n t of e x p e r i m e n t a l tissue was twice t h a t of the S T S - 3 e x p e r i m e n t a n d the m e a s u r i n g a n d s e c t i o n i n g were c o n d u c t e d at the l a n d i n g site. T h e two P G U ' s were received at the l a n d i n g site l a b o r a t o r y 2 hrs after t o u c h d o w n . G a s samples were t a k e n f r o m the P G C ' s a n d the p l a n t s were observed a n d p h o t o g r a p h e d . T h e seedlings were r e m o v e d , m e a s u r e d , w e i g h e d , s e c t i o n e d , a n d p l a c e d i n labeled containers. T h e tissues designated for l i g n i n a n a l y s i s were c h i l l e d w h i l e those to be used for e n z y m e a n a l y s i s were frozen on d r y ice. T h e tissues either chilled or frozen were r e t u r n e d to the P I l a b o r a t o r y where the l i g n i n s a m p l e s were i m m e d i a t e l y t r e a t e d w i t h 1.5 m l a c e t o n e / 1 % H C 1 as before. Control Experiments. T h e one g r a v i t y ( l g ) c o n t r o l for the S T S - 3 e x p e r i m e n t was c o n d u c t e d d u r i n g the flight m i s s i o n . T h e c o n t r o l P G C ' s were p r e p a r e d at the same t i m e as those designated for flight. I n fact, the c o n t r o l P G U was t r a n s p o r t e d to the l a u n c h p a d as a b a c k u p e x p e r i m e n t a l p a c k age b u t was r e t u r n e d to the o p e r a t i o n s b u i l d i n g w h e n the flight P G U was declared f u n c t i o n a l . T h e c o n t r o l P G U was connected to g r o u n d s u p p o r t e q u i p m e n t for power a n d m o n i t o r i n g . T h e l g c o n t r o l for the S T S - 5 1 F e x p e r i m e n t was c o n d u c t e d p o s t - f l i g h t at N A S A K S C L i f e S u p p o r t F a c i l i t i e s . T h e l o a d e d P G U ' s were connected to g r o u n d s u p p o r t e q u i p m e n t located i n a c o m p u t e r - c o n t r o l l e d e n v i r o n m e n t a l r o o m . T h e t e m p e r a t u r e of the r o o m was m a i n t a i n e d s u c h t h a t the P G U ' s were able to m a i n t a i n P G C t e m p e r a t u r e profiles s i m i l a r to those of flight. T h e flight t i m e profile also was s i m u l a t e d i n the c o n t r o l e x p e r i m e n t . Lignin Analysis L i g n i n q u a n t i t a t i o n was based u p o n a m o d i f i e d p r o c e d u r e of J o h n s o n , M o o r e , a n d Z a n k (7) a n d v a n Z y l (8). I n the S T S - 3 e x p e r i m e n t eight seedlings f r o m each P G C (every other seedling) were selected for l i g n i n a n a l y s i s . I n the S T S - 5 1 F e x p e r i m e n t , five seedlings f r o m each P G C were
208
PLANT C E L L W A L L P O L Y M E R S
selected for l i g n i n a n a l y s i s . T h e pine h y p o c o t y l s were cut i n t o s i x equal sections a n d oat stems i n t o two equal sections i n b o t h e x p e r i m e n t s . In the S T S - 3 e x p e r i m e n t o n l y three 1 c m m u n g b e a n stems were a n a l y z e d . These were the sections located j u s t below the needles a n d cotyledons a n d j u s t above the root c o l l a r . In the S T S - 5 1 F e x p e r i m e n t , m u n g beans were first cut i n t o h y p o c o t y l a n d e p i c o t y l regions a n d each region was further d i v i d e d i n t o three e q u a l sections. T h e lower oat s t e m was cut i n t o two 3 c m sections. E a c h s t e m section c o n s t i t u t e d a s a m p l e . Before digestion each section was cut i n t o a p p r o x i m a t e 2 m m subsections. T h e subsections were e x t r a c t e d i n a series of steps: 1.5 m l absolute a c e t o n e / 1 % H C 1 for 24 h r at - 1 0 ° C , 1.5 m l 2 N N a O H for 48 hr at r o o m t e m p e r a t u r e , a n d 7 m l deionized water for 24 hr at r o o m t e m p e r a t u r e . T h e samples were d e h y d r a t e d w i t h 0.5 m l acetone a n d oven-dried for 1 h r at 70°C. T h e samples were digested i n 0.4 m l 2 5 % a c e t y l b r o m i d e / g l a c i a l acetic a c i d for 1 hr at 70°C, a n d d i l u t e d w i t h 2.7 m l g l a c i a l acetic a c i d / N a O H (15:2.7) a n d 20 μ\ 7.5 M h y d r o x y l a m i n e - H C l . A f t e r s o l u b i l i z a t i o n the samples were q u a n t i t a t e d s p e c t r o p h o t o m e t r i c a l l y (A280) a n d c o m p a r e d to a w o o d l i g n i n s t a n d a r d . E n z y m e and Protein Analysis S i x pine seedlings f r o m each P G C were selected for enzyme a n d p r o t e i n analysis i n the S T S - 3 a n d S T S - 5 1 F e x p e r i m e n t s . T h e pine seedlings were sectioned as described p r e v i o u s l y for l i g n i n analysis. A l l s i x sections f r o m each region were pooled for a n a l y s i s . T h e samples were frozen at this p o i n t . L a t e r the samples were homogenized 30 to 60 sec at 4 ° C u s i n g a P o l y t r o n homogenizer. T h e tissues were homogenized i n 1.5 m l of buffer c o n t a i n i n g 0.1 M N a - b o r a t e buffer ( p H 8.8) c o n t a i n i n g 0.1 M K G a n d soluble p o l y v i n y l p y r r o l i d i n e ( P V P ) ( 2 0 % m g / m l ) . A f t e r c e n t r i f u g a t i o n for 10 m i n at 15,000 r p m the s u p e r n a t a n t s were transferred to ice-cold test tubes. P h e n y l a l a n i n e a m m o n i a lyase ( P A L ) a c t i v i t y was d e t e r m i n e d as p r e v i o u s l y described (9). T h e r e a c t i o n m i x t u r e contained 0.25 m l 0.4 M s o d i u m borate buffer ( p H 8.8) c o n t a i n i n g 0.4 M K C 1 , 0.1 m l 100 m M p h e n y l a l a n i n e , 0.1 m l 2 / i C i / m l ( U - C ) - p h e n y l a l a n i n e , 0.1 m l tissue homogenate a n d deionized H 2 O to a v o l u m e of 1.0 m l . T h e m i x t u r e was i n c u b a t e d 1 hr at 3 7 ° C a n d the r e a c t i o n s t o p p e d b y a d d i t i o n of 0.4 m l 5 0 % t r i c h l o r o a c e t i c acid ( T C A ) . Peroxidase a c t i v i t y was based o n the p o l y m e r i z a t i o n rate of g u a i a c o l i n the presence of hydrogen peroxide (10). T h e reaction m i x t u r e contained 2.0 m l 13 m M p o t a s s i u m phosphate buffer ( p H 7.0) c o n t a i n i n g 5 m M H 2 O 2 , 0.7 m l 15 m M g u a i a c o l , a n d 0.1 m l of tissue e x t r a c t . T h e reaction rates were m o n i t o r e d c o n t i n u o u s l y i n a spectrophotometer. C h a n g e s i n absorbance (A470) were recorded at 15 sec intervals for 2 to 3 m i n . P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d b y use of C o o m a s s i e B l u e reagent (11). 1 4
Results T h e p r i n c i p a l e x p e r i m e n t a l objective of the S T S - 3 a n d S T S - 5 1 F flight ex p e r i m e n t s was to determine whether y o u n g p l a n t seedlings g r o w n p r e d o m -
15.
COWLESETAL.
Seedlings Grown on Earth & on Space Shuttle
209
i n a n t l y i n a m i c r o g r a v i t y e n v i r o n m e n t w o u l d r e s p o n d b y s y n t h e s i z i n g less l i g n i n . T h e S T S - 3 e x p e r i m e n t was designed to be a p r e l i m i n a r y e x p e r i m e n t t h a t w o u l d p r o v i d e useful results i n p l a n n i n g the Spacelab II e x p e r i m e n t to be flown o n S T S - 5 1 F . It was i m p o r t a n t , however, t h a t at least some of the S T S - 5 1 F e x p e r i m e n t be a repeat of the S T S - 3 e x p e r i m e n t . T h i s was a c c o m p l i s h e d i n p r i n c i p l e i n t h a t (1) the same three p l a n t species were flown o n b o t h m i s s i o n s ; (2) d u r a t i o n s of the m i s s i o n s were s i m i l a r (194 hr for S T S - 3 a n d 190.5 h r for S T S - 5 1 F ) ; a n d (3) the same regions of oats a n d p i n e were a n a l y z e d i n the two e x p e r i m e n t s , w h i l e m o r e o f the m u n g bean s t e m was a n a l y z e d i n the S T S - 5 1 F e x p e r i m e n t . It is i m p o r t a n t , however, to note c e r t a i n m a j o r differences i n the t w o e x p e r i m e n t s . F i r s t l y , the l a m p s r e m a i n e d o n c o n t i n u o u s l y i n the S T S - 5 1 F e x p e r i m e n t r a t h e r t h a n b e i n g o n a 1 4 / 1 0 h r d a y / n i g h t cycle as w i t h the S T S - 3 e x p e r i m e n t . Secondly, the t e m p e r a t u r e i n the P G C ' s r e m a i n e d j u s t above 2 6 ° C i n the S T S - 5 1 F e x p e r i m e n t r a t h e r t h a n c y c l i n g between 23° C a n d 26° C w i t h the d a y / n i g h t c y c l i n g of the S T S - 3 e x p e r i m e n t . F i n a l l y , there were t w i c e as m a n y seedlings i n the S T S - 5 1 F e x p e r i m e n t . P i n e seedlings t h a t were 4 days o l d at l a u n c h a n d 12 days o l d at l a n d i n g were a n a l y z e d for l i g n i n i n b o t h flight e x p e r i m e n t s . I n b o t h cases, flight seedlings c o n t a i n e d less l i g n i n t h a n c o n t r o l seedlings ( T a b l e I). T h e average decrease i n l i g n i n i n flight seedlings over controls was 4 . 4 % a n d 5 . 5 % for the S T S - 3 a n d S T S - 5 1 F e x p e r i m e n t s , respectively. T h i s s m a l l difference i n l i g n i n content between flight a n d c o n t r o l seedlings was s t a t i s t i c a l l y s i g n i f i c a n t at the 0 . 5 % level i n the S T S - 5 1 F e x p e r i m e n t b u t not i n the S T S - 3 e x p e r i m e n t . In b o t h e x p e r i m e n t s the largest r e d u c t i o n i n pine seedling l i g n i n o c c u r r e d i n the u p p e r h y p o c o t y l region. T a b l e I. L i g n i n C o n t e n t i n 12-day-old P i n e Seedlings Flight Number STS-3 Section Number 1 2 3 4 5 6
STS-51F
Flight C o n t r o l Difference ( / i g / s t e m section) (%) 157.3 137.8 112.7 85.2 55.8 28.5
162.1 139.6 117.9 88.0 58.4 37.6 Av:
- 2.9 - 1.3 - 4.4 - 3.1 - 4.6 -24.2 -
4.4
Flight Control Difference ( ^ g / s t e m section) (%) 208.2 188.3 155.5 133.8 107.5 62.5
198.7 187.5 166.0 146.4 123.6 72.7 Av:
+ 4.8 + 0.4 - 6.3 - 8.6 -13.0 -14.0 -
5.5
T h e S T S - 5 1 F e x p e r i m e n t also i n c l u d e d p i n e seedlings t h a t were 10 d a y s o l d at l a u n c h a n d 18 days o l d at l a n d i n g . T h e l i g n i n content i n flight seedlings was r e d u c e d 1 3 . 0 % i n c o m p a r i s o n to controls ( T a b l e II). In t h i s
210
PLANT C E L L W A L L P O L Y M E R S
set of seedlings the r e d u c t i o n i n l i g n i n content was significant at the 0 . 1 % l e v e l . T h e r e d u c t i o n i n l i g n i n content i n older flight seedlings was more evenly d i s t r i b u t e d over the h y p o c o t y l t h a n i n the younger pine seedlings. T a b l e II. L i g n i n C o n t e n t i n 18-day-old P i n e Seedlings F l o w n o n S T S - 5 1 F Flight Control ( / i g / s t e m section)
Section Number 1 2 3 4 5 6
162.7 173.3 154.9 141.5 118.8 79.4
Difference (%)
205.5 197.9 179.2 153.5 128.9 89.5
-20.8 -12.4 -13.6 - 7.8 - 7.8 -11.3
Av:
-13.0
M u n g beans were s t a r t e d as seeds i n the S T S - 3 a n d S T S - 5 1 F e x p e r i m e n t s a n d g e r m i n a t e d a n d g r o w n to a n average height o f a b o u t 12 c m w i t h i n the 8-day e x p e r i m e n t a l periods. In the S T S - 3 e x p e r i m e n t o n l y three 1 c m sections of m u n g b e a n stems were a n a l y z e d for l i g n i n . T h e s e w o u l d be e q u i v a l e n t t o a p o r t i o n o f sections 3, 4 a n d 6 i n the S T S - 5 1 F e x p e r i m e n t ( T a b l e I I I ) . F l i g h t m u n g b e a n seedlings i n the S T S - 3 e x p e r i m e n t c o n t a i n e d 1 6 % less l i g n i n t h a n c o n t r o l seedlings. T h i s r e l a t i v e l y large a n d s t a t i s t i c a l l y significant difference i n l i g n i n content between flight a n d c o n t r o l m u n g beans led to a more extensive a n a l y s i s of m u n g beans i n the S T S - 5 1 F e x p e r i m e n t . T h e results f r o m the l a t t e r e x p e r i m e n t showed a n even greater r e d u c t i o n (37%) i n l i g n i n i n flight as c o m p a r e d to c o n t r o l seedlings. T h e largest change i n l i g n i n content i n the m i c r o g r a v i t y e n v i r o n m e n t was i n the younger s t e m region. T a b l e I I I . L i g n i n C o n t e n t i n M u n g B e a n Seedlings Flight Number STS-3 Section Number 1 2 3 4 5 6
STS-51F
Flight C o n t r o l Difference ( ^ g / s t e m section) (%) 50.6
59.6
-15.1
32.6
41.9
-22.2
17.2
21.0
-18.2
Av:
-16.1
—
— —
— — —
—
— —
Flight Control Difference (/L/g/stem section) (%) 138.6 116.1 109.3 131.0 77.3 42.5
186.2 160.4 167.7 235.4 162.6 69.9
-25.6 -27.6 -34.8 -44.4 -52.5 -39.2
Av:
-37.4
15.
COWLESETAL
Seedlings Grown on Earth & on Space Shuttle
111
S i x 12-day-old p i n e seedlings p e r P G C were u t i l i z e d to d e t e r m i n e p r o tein content a n d P A L a n d peroxidase a c t i v i t y i n b o t h the S T S - 3 a n d S T S 5 1 F e x p e r i m e n t s . I n b o t h e x p e r i m e n t s , flight seedlings contained signifi c a n t l y less P A L a c t i v i t y t h a n c o n t r o l seedlings ( T a b l e I V ) . T h e average r e d u c t i o n i n P A L a c t i v i t y was 2 2 . 2 % for the S T S - 3 e x p e r i m e n t a n d 1 8 . 3 % for t h e S T S - 5 1 F e x p e r i m e n t . T h e change i n P A L a c t i v i t y was more or less evenly d i s t r i b u t e d a l o n g the pine h y p o c o t y l . T a b l e I V . P A L A c t i v i t y i n 12-day-old P i n e Seedlings Flight Number STS-3 Section Number 1 2 3 4 5 6
STS-51F
Flight C o n t r o l Difference ( ^ g / s t e m section) (%) 3.78 5.44 5.88 6.58 6.15 3.67
5.25 6.30 7.28 7.51 8.62 5.53
-28.0 -13.6 -19.3 -12.4 -28.7 -33.6
AV:
-22.2
Flight C o n t r o l Difference ( / i g / s t e m section) (%) 3.27 3.62 4.16 4.45 4.51 3.05
4.67 4.07 5.73 5.17 5.62 2.95
-30.0 -11.1 -27.4 -13.9 -19.8 + 3.4
AV:
-18.3
P e r o x i d a s e a c t i v i t y i n flight seedlings was reduced to essentially the same extent as P A L a c t i v i t y . A g a i n , the average difference i n the S T S - 3 ( - 1 9 . 0 % ) a n d S T S - 5 1 F ( - 2 0 . 2 % ) e x p e r i m e n t s were s i m i l a r ( T a b l e V ) . A s w i t h P A L a c t i v i t y the r e d u c t i o n i n peroxidase a c t i v i t y i n flight seedlings was s i m i l a r a l o n g the pine h y p o c o t y l . T a b l e V . Peroxidase A c t i v i t y i n 12-day-old P i n e Seedlings Flight Number STS-3 Section Number 1 2 3 4 5 6
STS-51F
Flight C o n t r o l Difference (/zg/stem section) (%)
Flight C o n t r o l Difference ( ^ g / s t e m section) (%)
47.96 42.56 45.56 41.76 42.91 56.41
68.61 60.95 55.27 47.36 49.13 42.68
59.16 57.06 53.91 50.06 52.86 68.96
-18.9 -25.4 -15.5 -16.6 -18.8 -18.2
Av:
-19.0
107.75 72.12 61.29 56.85 55.26 52.81
-36.3 -15.5 - 9.8 -16.7 -11.1 -19.2
Av:
-20.2
212
PLANT C E L L W A L L POLYMERS
Discussion O u r c o n c l u s i o n is t h a t y o u n g higher p l a n t seedlings perceive m i c r o g r a v i t y a n d r e s p o n d b y s y n t h e s i z i n g s i g n i f i c a n t l y less l i g n i n . L i g n i n content was r e d u c e d i n a l l seven sets o f flight a n d c o n t r o l seedlings c o m p a r e d i n the two e x p e r i m e n t s . In five sets of seedlings, two m u n g beans, two pines a n d one o a t , the difference i n l i g n i n between flight a n d c o n t r o l e x p e r i m e n t s r a n g e d f r o m 6 to 2 4 % a n d a l l were s t a t i s t i c a l l y s i g n i f i c a n t . T h e t w o sets of seedlings, oats a n d 4 - d a y - o l d pines t h a t e x h i b i t e d s m a l l e r a n d s t a t i s t i c a l l y insignificant differences i n l i g n i n were p a r t of the S T S - 3 e x p e r i m e n t where the t o t a l a m o u n t of l i g h t received was considerably less t h a n i n the S T S - 5 1 F e x p e r i m e n t . B o t h o f these seedling types e x h i b i t e d a significant difference i n l i g n i n i n the S T S - 5 1 F e x p e r i m e n t . T h e m a g n i t u d e o f the r e d u c t i o n i n l i g n i n content due to m i c r o g r a v i t y was especially significant considering t h a t (1) l i g n i f i c a t i o n is a d e v e l o p m e n t a l process; (2) the e x p e r i m e n t a l p e r i o d was r e l a t i v e l y short for d e m o n s t r a t i n g differences i n l i g n i f i c a t i o n ; (3) the e x p e r i m e n t a l tissue was not a p p r e c i a b l y w e i g h t - b e a r i n g ; a n d (4) the m i c r o g r a v i t y e n v i r o n m e n t of the s h u t t l e was i n the 1 χ 1 0 ~ g range, w h i c h is not near weightlessness. 3
T h e results on P A L a n d peroxidase a c t i v i t y also s u p p o r t the hypothesis t h a t y o u n g seedlings perceive m i c r o g r a v i t y a n d synthesize less l i g n i n . T h e a c t i v i t y of b o t h enzymes was reduced about 2 0 % i n flight seedlings over controls i n b o t h e x p e r i m e n t s . These results were very consistent a n d were s t a t i s t i c a l l y s i g n i f i c a n t . W h e t h e r reduced a c t i v i t y of these enzymes was p r i n c i p a l l y responsible for reduced l i g n i n content r e m a i n s to be d e t e r m i n e d . T h e e x p e r i m e n t a l procedures a n d assays were designed to measure t o t a l P A L a n d peroxidase a c t i v i t y a n d not j u s t t h a t associated w i t h l i g n i f i c a t i o n . T h e s e same procedures a n d assays, however, have been used to d e m o n s t r a t e a p o s i t i v e c o r r e l a t i o n between the a c t i v i t y o f these enzymes a n d l i g n i f i c a t i o n d u r i n g e l o n g a t i o n i n y o u n g pine seedlings (increased e n z y m e a c t i v i t y at the t i m e of increased l i g n i f i c a t i o n , u n p u b l i s h e d results). T h e results between the two flight e x p e r i m e n t s were p o s i t i v e l y related or c o u l d be accounted for by k n o w n e x p e r i m e n t a l differences. T h e difference between l i g n i n content of 12-day-old flight a n d c o n t r o l pine seedlings was s i m i l a r between the two e x p e r i m e n t s , as were P A L a n d peroxidase a c t i v i t y . T h e significant r e d u c t i o n i n l i g n i n content i n flight m u n g beans a n d oats i n the S T S - 5 1 F e x p e r i m e n t , as c o m p a r e d t o the S T S - 3 e x p e r i m e n t , was p r o b a b l y due to the p o s i t i v e response of these p l a n t species to the a d d i t i o n a l a m o u n t of light a n d to the s l i g h t l y higher t e m p e r a t u r e s of the S T S - 5 1 F e x p e r i m e n t . T h e a m o u n t of l i g n i n i n b o t h flight a n d c o n t r o l seedlings was higher i n the S T S - 5 1 F e x p e r i m e n t (Tables I a n d III). A g a i n , t h i s was p r o b a b l y a p o s i t i v e response to the increased a m o u n t of l i g h t i n the S T S - 5 1 F e x p e r i m e n t . T h e difference i n light also helped e x p l a i n the d a t a s h o w n i n T a b l e III where the a m o u n t of l i g n i n i n the S T S - 5 1 F e x p e r i m e n t was 2 to 4 t i m e s t h a t of the S T S - 3 e x p e r i m e n t . A n o t h e r i m p o r t a n t difference i n the m u n g bean d a t a was t h a t the S T S - 3 values for b o t h the flight a n d c o n t r o l seedlings were for 1 c m sections a n d the S T S - 5 1 F values were o n e - s i x t h of the s t e m l e n g t h , w h i c h averaged more t h a n 2 c m .
15.
COWLES ET AL.
Seedlings Grown on Earth & on Space Shuttle
213
It is i m p o r t a n t t h a t these flight e x p e r i m e n t s be followed b y exposure of p l a n t s t o longer e x p e r i m e n t a l periods i n m i c r o g r a v i t y . W h i l e results r e p o r t e d here suggest a significant r e d u c t i o n i n l i g n i f i c a t i o n as a result o f m i c r o g r a v i t y , other experiments are needed t o establish (1) the extent o f r e d u c t i o n over t i m e ; (2) h o w different levels o f m i c r o g r a v i t y effect l i g n i fication; a n d (3) h o w r e d u c t i o n i n l i g n i f i c a t i o n is b r o u g h t a b o u t at t h e b i o c h e m i c a l a n d cellular level. A cknowledgment s W e w i s h t o t h a n k D r . T h o r a H a l s t e a d , N A S A headquarters, for her e x t r a o r d i n a r y support for the o v e r a l l project, D r s . E d M e r e k a n d J o h n T r e m o r , N A S A - A m e s , for their assistance as project scientists, a n d M r . R o n a l d M a n c i n i , Ν A S A - A m e s , for h a r d w a r e engineering. W e also acknowledge t h e capable assistance o f astronauts G o r d o n F u l l e r t o n a n d S t o r y M u s g r a v e , flight p a y l o a d manager M r . M a r t y E i b a n d , N A S A - G o d d a r d , a n d the 50 o r m o r e i n d i v i d u a l s at the N A S A centers w h o c o n t r i b u t e d t o the p r o j e c t . W e also t h a n k D r . Reff O m e n a n d M a r i l y n Stevens for their v a l u a b l e t e c h n i c a l a n d clerical assistance. T h e project was s u p p o r t e d i n p a r t b y N A S A g r a n t N S G 9042 a n d Ν A S 2-11165.
Literature Cited 1. Scott, D. R. M.; Preston, S. B. For. Sci. 1955, 1, 179-82. 2. Siegel, S. M.; Carrol, P.; Umeus, I.; Carre, C. Rec. Adv. Phytochem. 1972, 4, 223-38. 3. Scarfield, G. Science 1973, 179, 647-55. 4. Siegel, S.; Siegel, B.; Chen, J. In Life in the Universe; Billingham, J., Ed.; MIT: Boston, 1981; pp. 307-16. 5. Westing, A. H. Bot. Rev. 1968, 34, 51-78. 6. Cowles, J. R.; Scheld, H. W.; LeMay, R.; Peterson, C. Ann. Bot. Suppl. 1984, 3, 33-48. 7. Johnson, D. B.; Moore, W. E.; Zank, L. C. Tappi 1961, 44, 793-98. 8. van Zyl, J. D. Wood Sci. Technol. 1978, 12, 251-59. 9. Lau, Y. L.; Scheld, H. W.; Cowles, J. R. Physiol. Plant. 1980, 49, 299-303. 10. Chance, B.; Machly, A. C. In Methods in Enzymology; Colowick, S. P.; Kaplan, N. O., Eds.; Academic: New York, 1955; Vol. II, pp. 764-73. 11. Bradford, M. M. Anal. Biochem. 1976, 72, 248-54. RECEIVED May 19, 1989
