Research Note

Changes in isoenzymes and amino acids in forageand germination of the ®rst post-¯ight generationof seeds of three legume species after space ¯ight

Y. Y. Xu, J. F. Jia, J. B. Wang and B. T. Niu

Cell Biology Laboratory, Lanzhou University, PR China

Abstract

Seeds of sainfoin (Onobrychis viciifolia Scop.), alfalfa

(Medicago sativa L.) and Astragalus adsurgens Pall. were

put into and recovered from earth orbit from China in

1994. The isoenzymes in leaves and ¯owers and amino

acids in leaves of the ®rst post-¯ight generation of

plants were analysed. Germination of their seeds under

NaCl and polyethylene glycol (PEG) stress were tested.

The results showed that space ¯ight caused some

changes in the pattern of peroxidase and esterase in

sainfoin and A. adsurgens, and variation in amylase

patterns in leaves of alfalfa. The total amino acid

content increased in sainfoin and alfalfa leaves. Under

salt and water stress, the proportion of progeny seeds of

alfalfa and A. adsurgens, respectively, germinating after

7 and 18 days' imbibition was not markedly different

between treatment and control. However, the progeny

seeds of sainfoin which had been in orbit exhibited

higher tolerance to NaCl and PEG stress during germi-

nation1 .

Introduction

Since plants can serve as a central component for a

controlled ecological life support system for human

habitation in space, the responses of higher plants to

space conditions have been explored during the past

three decades (Merkys et al., 1984; Halstead and Dut-

cher, 1987). Both cosmic radiation and microgravity are

important factors affecting plants in space (Merkys

et al., 1984). Cosmic radiation induced changes in

chromosomal DNA in plants recovered from space

¯ight (Li et al., 1996). Although some aspects of the

plant life cycle, e.g. the development and differentiation

of tissue (Kuang et al., 1996), have appeared normal

under space conditions, other changes have been

observed. These include reduction in lignin and cellu-

lose content, peroxidase and phenylalanine ammonia

lyase activity (Cowles et al., 1984), alteration in cell

shape and subcellular structure (Halstead and Dutcher,

1987), an increase in frequency of chromosome aber-

ration and gene mutation (Kostina et al., 1984) and a

decrease in growth rate (Cowles et al., 1984) and

phenotypic changes (Mei et al., 1996).

Although basic research in cosmic radiation is pri-

marily concerned with the protection of both humans

and plants from the harmful effects of space, unpro-

tected space ¯ight of seeds could be advantageous in

producing useful genetic mutation. Based on this

concept, some mutant lines with favourable characters,

such as early maturity, dwarf, high yield and disease

resistance, have been obtained from seeds carried by

recoverable satellite (Jiang, 1996).

Sainfoin (Onobrychis viciifolia Scop.), alfalfa (Medicago

sativa L.) and Astragalus adsurgens Pall. are all perennial

legume forages. These are very nutritious to livestock,

and the plants can be utilized as windbreaks or for sand-

®xation, desalination and improving soil. To investigate

whether the genetic changes, described above, included

effects on isoenzymes and amino acids and the ability of

plants to withstand stress, changes in isoenzymes and

amino acid content in plants of the ®rst post-¯ight

generation and the sensitivity of the seeds of the

offspring to salt and water stress were evaluated.

Materials and methods

Plant materials

Air-dried seeds of sainfoin (cv. Greatwall No.1) were

provided by the Department of Grassland Science,

Gansu Agricultural University, China. The seeds of

alfalfa (cv. Gannong No.1) and A. adsurgens (cv.

Huanghe No.2) were obtained from the Grassland

Ecology Research Institute of Gansu province, China.

Seeds (20 g) of each species (Treatment SP1), packed in

Correspondence to: Professor J. F. Jia, Cell Biology Labo-

ratory, Lanzhou University, Lanzhou, 730000, PR China.

E-mail: xuyy@lzu.edu.cn

Received 4 September 1998; revised 24 April 1999

Ó 1999 Blackwell Science Ltd. Grass and Forage Science, 54, 371±375 371

cloth bags, were carried in recoverable satellite No.

940703, which was launched on 3 July 1994 and was

recovered on 18 July 1994. The space ¯ight lasted for 14 d

19 h. The ¯ight height was 175 to 350 km, the inner

temperature was ±20 to 35°C, the microgravity level

10±5 ´ g, the radiation level was 12á8 mGy d±1 and the

vacuum level was 10±8 Pa. The uncarried seeds (CK1)

were stored at ±20°C for 7 d and then at room temper-

ature until the satellite was recovered. After space ¯ight,

both SP1 and CK1 seeds were stored at 4°C. In 1995,

both SP1 and CK1 were sown in rows (ten plants in each

row) in the ®eld (36°02¢46¢¢N, 103°51¢35¢¢E) for the

production of a subsequent generation of seeds (SP2 and

CK2) and 100 plants of each species were planted in

plots of 50 m2. In 1996, the leaves and ¯owers at the

¯owering stage were collected and the seeds were

harvested from representative plants, grown from seed

in 1995, for the following experiments.

Isoenzyme analyses

The fresh leaves (5 g) and fresh ¯owers (3 g) were

collected randomly from different plants at the same

stage of ¯owering and the fresh materials were homo-

genized in a mortar in homogenizing medium

(0á0625 mol l±1 Tris-HCl buffer, pH 6á7, containing

10% sucrose), with a ratio of 3 ml of homogenizing

medium to 1 g of leaves or 5 ml to 1 g of ¯owers.

Homogenate was centrifuged for 5 min at 104 ´ g at

4°C, and 15 ll of supernatant was used for electropho-

resis on discontinuous polyacrylamide gel.

To prepare 10 ml of 10% separating gel, 1á25 ml of

separating gel buffer (3 mol l±1 Tris-HCl, pH 8á9, con-

taining 0á23% N,N,N¢,N¢-tetramethylethylenediamine),

3á3 ml of stock solution for separating gel (30% acryl-

amide±0á8% methylenebisacrylamide), 1á25 ml of 0á56%

ammonium persulphate and 4á2 ml of distilled water

were mixed together. Stacking gel (4%) consisted of one

part of stacking gel buffer (0á5 mol l±1 Tris-HCl, pH 6á7,

containing 0á46% N,N,N¢,N¢-tetramethylethylenedi-

amine), two parts of stock solution for stacking gel

(10% acrylamide±2á5% methylenebisacrylamide), one

part of 0á56% ammonium persulphate and four parts of

distilled water. The electrophoresis buffer was composed

of 5 mmol l±1 Tris and 38 mmol l±1 glycine (pH 8á3).

Separation of esterase isoenzymes was performed on

10% acrylamide gel and peroxidase and amylase iso-

enzymes on 6% acrylamide gel. The gels were all run at

4°C and under non-denaturing conditions. The perox-

idase isoenzymes were detected on gel using benzidine

and H2O2 as substrate, and the esterase isoenzymes were

detected using 1-naphthyl acetate and 2-naphthyl ace-

tate as substrate. To detect the amylase, the gel was

incubated in 1% soluble starch solution for 30 min after

which the gel was transferred to the I2±KI solution until

the transparent bands appeared against the blue back-

ground. The gels were photographed after staining.

Amino acid analyses

Leaves of plants were dried at 80°C for 48 h. Samples

(150 mg) were hydrolysed in 6 N HCl at 110°C for 22 h,

Figure 1 Electrophoresis separations. (a±c)

Peroxidase on 6% polyacrylamide gels; (d±f)

esterase on 10% polyacrylamide gels; (g±i)

amylase on 6% polyacrylamide gels. Arrows

indicate the difference in band patterns

between space ¯ight recovered (SP1, lane 2

and lane 4) and control (CK1, lanes 1 and 3)

seeds. Lanes 1 and 2 are results for leaves

and lanes 3 and 4 those for ¯owers. Patterns

for sainfoin are shown in a, d and g, those for

alfalfa in b, e and h, and for Astragalus

adsurgens in c, f and i.

372 Y. Y. Xu et al.

Ó 1999 Blackwell Science Ltd, Grass and Forage Science, 54, 371±375

and the amino acids were determined with an Hitachi

835-50 high-speed amino acid analyser (Hitachi, Tokyo,

Japan).

Germination tests of progeny seedsunder salt and water stresses

SP2 and CK2 seeds, collected in 1996, were placed in

9-cm Petri dishes containing two sheets of ®lter paper

saturated with 0%, 0á6%, 1á2%, 1á5% and 1á8% NaCl

or 0%, 10%, 20%, 25% and 30% polyethylene glycol

(PEG, MW 6000) solution. Germination tests were

carried out in the controlled environment cabinets at

25 � 1°C under 1200 lux of light. The germination

proportions of SP2 and CK2 seeds were calculated

under each stress condition.

Statistical analysis

The difference between means of total amino acid

content in leaves between SP1 and CK1 was examined

by the t-test. To evaluate the effect of space ¯ight on salt

and water stress resistance, the germination proportion

of SP2 seeds and CK2 seeds under the same stress

conditions were compared according to the formula,

d� (k1 ± k2)/Ö[k(1 ± k)(1/n1 + 1/n2)] given by Bailey

(1959), where k1� germination proportion of SP2 seeds,

k2� germination proportion of CK2 seeds and k�germination proportion of total of SP2 and CK2 seeds.

Results and discussion

Isoenzymes

Isoenzymes, as one of the products of gene expression,

have often been used as markers in genetics to study

development, differentiation and plant variation in

breeding. The changes of isoenzyme patterns could

re¯ect gene expression or even gene changes. Cowles

et al. (1984) reported that changes of enzyme activity

were found in seedlings exposed in space, and after

space ¯ight the damage that resulted from gene muta-

tion can be preserved at least in the ®rst post-¯ight

generation (Kostina et al., 1984). In the current work,

changes in isoenzymes were found in plants of three

forage legumes 2 years after sowing seeds previously

subjected to space ¯ight.

In sainfoin, Figure 1a shows differences in peroxidase

isoenzyme content between SP1 and CK1: two bands

(marked by arrows) present in CK1 ¯owers (Figure 1a,

lane 3) were absent in SP1 ¯owers (Figure 1a, lane 4).

The pattern of peroxidase isoenzymes in leaves and

Table 1 The content of amino acids (g amino acid 100 g)1 dry weight) in leaves of three forage legumes grown from seeds subjected to space ¯ight

(SP1) compared with untreated controls (CK1). Values of the total amino acid content are the mean of three replicates with standard error in

brackets.

Sainfoin Alfalfa Astragalus adsurgens

Amino acid SP1 CK1 SP1 CK1 SP1 CK1

Aspartate 2á95 1á87 3á36 2á42 2á38 2á39

Threonine 1á46 0á99 1á49 1á25 1á21 1á21

Serine 1á28 0á91 1á37 1á16 1á10 1á14

Glutamate 3á18 2á25 3á31 2á88 2á68 2á65

Glycine 1á54 1á07 1á56 1á35 1á26 1á26

Alanine 1á94 1á23 1á95 1á58 1á49 1á49

Valine 1á71 1á19 1á76 1á49 1á35 1á36

Methionine 0á27 0á30 0á28 0á22 0á18 0á25

Isoleucine 1á30 0á92 1á35 1á17 1á10 1á09

Leucine 2á48 1á82 2á56 2á30 2á08 2á08

Tyrosine 1á07 0á75 1á10 0á95 0á90 0á97

Phenylalanine 1á66 1á14 1á68 1á41 1á35 1á35

Lysine 1á86 1á35 1á91 1á66 1á53 1á58

Histidine 0á67 0á47 0á67 0á60 0á54 0á55

Arginine 1á66 1á19 1á77 1á51 1á41 1á40

Proline 1á52 0á93 1á52 1á17 1á16 1á16

Cysteine 0á57 0á35 0á59 0á42 0á45 0á49

Tryptophan 0á25 0á19 0á54 0á44 0á51 0á35

Total 27á37 (1á61)* 18á92 (1á78) 28á77 (2á01) 23á98 (1á51) 22á68 (1á46) 22á77 (1á80)

*Signi®cant difference between SP1 and CK1 of sainfoin at P < 0á05 according to t-test.

Isoenzymes, amino acids and germination changes in forage legumes after space ¯ight 373

Ó 1999 Blackwell Science Ltd, Grass and Forage Science, 54, 371±375

¯owers of SP1 alfalfa and SP1 A. adsurgens was almost

the same as that in CK1 (Figure 1b and c).

The change in esterase isoenzymes as a result of space

¯ight was as marked as peroxidase. There were addi-

tional bands in SP1 sainfoin leaves (Figure 1d, lane 2)

and a band absent in SP1 A. adsurgens ¯owers (Figure 1f,

lane 4). The esterase patterns in alfalfa did not change

after space ¯ight (Figure 1e).

In sainfoin and A. adsurgens leaves amylase patterns

were not changed by space ¯ight (Figure1g and i). In

alfalfa leaves, however, SP1 leaves did not have a high

activity band of amylase (Figure 1h, lane 1).

These correspond to the changes in esterase and

peroxidase bands found in the immature ears of wheat

grown on earth from ¯own seeds exposed to space

¯ight (Li et al., 1996). Therefore these data suggest that

isoenzyme can be in¯uenced by space ¯ight in the ®rst

post-¯ight generation.

Amino acid analyses

One of the purposes of forage breeding is to raise amino

acid content in leaves. The results in Table 1 indicate

that the total amino acid content in SP1 leaves of

sainfoin and alfalfa plants was increased by space ¯ight

when compared with plants from the untreated control,

but this did not occur in A. adsurgens leaves. Only in the

case of methionine in the SP1 treatment was the amino

acid content less in sainfoin leaves; this showed a

decrease to 90% of that in CK1 leaves.

Table 2 Effects of NaCl polyethylene glycol (PEG) stress on germination proportion of seeds of sainfoin and alfalfa after imbibition for 7 days

and of Astragalus adsurgens after imbibition for 18 days.

Sainfoin

NaCl concentration (%) PEG concentration (%)

0 0á6 1á2 1á5 1á8 0 10 20 25 30

SP2 total seeds 195 90 90 90 90 126 60 60 60 60

Germination proportion

(s.e.)

0á84

(0á026)

0á60

(0á051)

0á17

(0á039)

0á06

(0á025)

0á01

(0á01)

0á80

(0á036)

0á75

(0á056)

0á78

(0á053)

0á50

(0á065)

0á15

(0á046)

CK2 total seeds 195 90 90 90 90 60 60 60 60 60

Germination proportion

(s.e.)

0á78

(0á030)

0á56

(0á052)

0á08

(0á029)

0 0 0á77

(0á054)

0á75

(0á056)

0á53

(0á064)

0á30

(0á059)

0á017

(0á017)

Signi®cance of difference between SP2 and CK2 ** * **

Alfalfa

NaCl concentration (%) PEG concentration (%)

0 0á6 1á2 0 10 20 25 30

SP2 total seeds 150 150 150 140 200 300 300 300

Germination proportion

(s.e.)

0á18

(0á031)

0á06

(0á019)

0 0á17

(0á032)

0á11

(0á022)

0á023

(0á009)

0á006

(0á004)

0

CK2 total seeds 150 150 150 300 300 300 300 300

Germination proportion

(s.e.)

0á13

(0á027)

0á04

(0á016)

0 0á18

(0á022)

0á15

(0á021)

0á023

(0á009)

0á01

(0á006)

0

Astragalus adsurgens

NaCl concentration (%)

0 0á6 0á8 1á0

SP2 total seeds 75 94 83 97

Germination proportion

(s.e.)

0á80

(0á046)

0á17

(0á039)

0á16

(0á027)

0á021

(0á015)

CK2 total seeds 80 73 42 67

Germination proportion

(s.e.)

0á84

(0á041)

0á14

(0á041)

0á10

(0á046)

0á015

(0á014)

SP2, ®rst generation post-¯ight seeds; CK2, ®rst generation untreated control seeds.

*P < 0á05; **P < 0á01 by the signi®cance test given by Bailey (1959) under the same PEG concentration.

374 Y. Y. Xu et al.

Ó 1999 Blackwell Science Ltd, Grass and Forage Science, 54, 371±375

Effects of NaCl and PEG stresson SP2 seed germination

Another purpose of plant breeding is to obtain stress-

resistant lines. In seedlings of asparagus grown from

¯own seeds, increased proline content and decreased

permeability of the plasma membrane were found by

Liu et al. (1993), and it was considered that these

seedlings had a stronger tolerance to salt stress.

Table 2 shows that the proportion of the ®rst-gener-

ation post-¯ight seeds of sainfoin (treatment SP2)

germinating in the presence of NaCl was higher than

that of CK2. For example, the proportion of SP2 seeds

germinating was 0á17 and that of CK2 was 0á08 in 1á2%

NaCl, and SP2 seeds of sainfoin exhibited resistance to

PEG stress (Table 2). For example, when the PEG

concentration was increased to 20%, the inhibition of

SP2 seeds by PEG was remarkably lower than that of

CK2 seeds; the proportion of SP2 seeds germinating was

signi®cantly greater (P < 0á01) than in CK2, i.e. 0á78

compared with 0á53.

The tolerances to salt and PEG stress were not

enhanced in offspring seeds of alfalfa (Table 2).

The inhibition of NaCl on the germination of both

SP2 and CK2 seeds of A. adsurgens was not markedly

different (Table 2).

Space ¯ight increased salt and water stress resistance

in SP2 sainfoin and A. adsurgens seeds and total content

of amino acids in sainfoin and alfalfa leaves, although

the reason for these changes being induced is not clear,

nevertheless these data suggest space ¯ight provides a

new potential method for inducing variation for forage

breeding.

References

BAI LEYAILEY N.T.J. (1959) Statistical Methods in Biology. London:

The English Universities Press.

COWLESOWLE S J.R., SC HE LDCHELD H.W., LEMAYE MAY R. and PETERSONETE RSON, C.

(1984) Growth and ligni®cation in seedlings exposed to

eight days of microgravity. Annals of Botany, 54 (Suppl.

3), 33±48.

HALSTEADALSTEAD T.W. and DUTCHERUTCHER F.R. (1987) Plants in space.

Annual Review of Plant Physiology, 38, 317±345.

JIANGIANG X.C. (1996) Development and prospect of space

mutation breeding in China. Chinese Journal of Space

Science, 16(suppl.), 77±82.

KOSTINAOSTI NA L., ANIKEEVANI KEE VA I. and VAULINAAULINA E. (1984) The

in¯uence of space ¯ight factors on viability and mut-

ability of plant. Advances in Space Research, 4, 65±70.

KUANGUANG A., XIAOIAO Y. and MUSGRAV EUSGRAVE M.E. (1996) Cyto-

chemical localization of reserves during seed develop-

ment in Arabidopsis thaliana under space ¯ight

conditions. Annals of Botany, 78, 343±351.

LII J.G., JIANGI AN G X.C. and WANGANG C.C. (1996) Effect of space

condition on the cytological characteristics and isozymes

of some cereal crops. Acta Genetica Sinica, 23, 48±55.

LIUI U C.D., SHENHE N Q.G., DUU X.G. and JUU R. (1993) Changes

in growth, development and physiological and bio-

chemical activity of asparagus seedlings grown from

space ¯own seeds. Acta Botanica Sinica, 35, 664±668.

ME IEI M.T., QZUZU YII ., HEE YII ., BUCKERUC KE R H. and YANGANG C.H.

(1996) Mutational effects of space ¯ight on Zea mays

seeds. Advances in Space Research, 14, 33±39.

ME RKY SERKYS A.J., LAURI NAVIC IUSAURINAV ICIUS R.S. and SVEGZDIENEVE GZDIE NE D.V.

(1984) Plant growth, development and embryogenesis

during Salyut-7 ¯ight. Advances in Space Research, 4,

55±63.

Isoenzymes, amino acids and germination changes in forage legumes after space ¯ight 375

Ó 1999 Blackwell Science Ltd, Grass and Forage Science, 54, 371±375