- Email: [email protected]
Influence of X-irradiation and seed-moisture on nucleic-acid and protein metabolism in barley
Radiation Botany, 1970, Vol. 10, pp. 437 to 443. Pergamon Press. Printed in Great Britain.
INFLUENCE OF X - I R R A D I A T I O N AND SEED-MOISTURE ON NUCLEIC-ACID AND P R O T E I N METABOLISM IN BARLEY* R. K. JOSHI Biology Division, Bhabha Atomic Research Centre, Trombay, Bombay-85, India ,*rid L. L E D O U X Biochimie Cellulaire, D~partment de Radiobiologie, Centre d'Etude de l'Energie Nucl~aire, Mol, Belgium
(Received 6 February 1970) Abstract---Seeds with varying moisture content (3-14 per cent) were X-irradiated with doses of 0, 10, 12-5 and 15 kR. Radiation-induced changes in the length of root and shoot, and their fresh and dry weights indicate that the seeds show maximum radioresistance at intermediate moisture levels and that the shoot is more sensitive to X-rays than the root. There was no significant modification of DNA, RNA and acid soluble (AS) content while protein content was adversely affected by radiation irrespective of seed moisture. However, no change in total macromolecular content could be detected in the plant as a whole. R~smm~----Des graines poss~dant des teneurs en eau vari~es (3 ~ 14%) ont ~tfi irradi~es par les rayons X aux doses de 0, 10, 12,5 et 15 kR. Les changements de longueur des racines et des apex induits par les radiations, ainsi que les poids frais et secs indiquent que les graines montrent un maximum de radio-r~sistance ~t des niveaux d'hydratation interm~diaires et que l'apex est plus sensible que la racine. I1 n'y a pas de modification significative des contenus en DNA, R N A ainsi qu'en acido-soluble alors que le contenu en protdines est invers~ment influenc~ par les rayons ind~pendamment de l'hydratation. On n'a cependant pu d~tecter de changement en contenu total des macromolficules des plantes enti6res. Z u s a m m e n f a s s u n g - - S a m e n mit verschiedenem Gehalt an Feuchtigkeit (3 bis 14%) wurden mit R~ntgenstrahlen yon 0, 10, 12,5 und 15 kR behandelt. Strahleninduzierte Ver~inderungen der L/inge der Wurzeln und Sch6sslinge und ihr Frisch- und Trockengewicht zeigen, dass die Samen den h6chsten Grad an Strahlenresistenz bei mittlerem Feuchtigkeitsgehalt aufweisen, und dass die Sch6sslinge gegeniiber den R6ntgenstrahlen empfindlicher sind als die Wurzeln. Es trat keine signifikante Modifikation der DNS, RNS und des s~iurel6slichen Gehaltes auf, dagegen wurde der Proteingehalt v o n d e r Strahlung beeinflusst ungeaehtet der Feuchtigkeit der Samen. Hinsichtlich des gesamten makromolikularen Gehaltes konnte aber in der Pflanze als ganze keine Ver~inderung festgestellt werden.
* This work was carried out at Section de Biochimie Cellulaire, D6partment de Radiobiologie, Centre d'Etude de l'Energie Nucl6aire, Mol, Belgium. 437
R. K. JOSItI and L. LEDOUX
438 INTRODUCTION
THE EFFECTS of sparsely ionizing radiations on seeds, in relation to their moisture content have been extensively studied (CALD~.COT'r,(3,4) BmBL and PAP~.(2)). The radiosensitivity of seeds varies greatly even with a little change in seed moisture (CoNOER et al.(6)). According to NOTANI and GAUR03) it appears to be minimal at intermediate moisture content and enhanced above and below this range. Such a relationship is oxygen-dependent (CoNOER el al.(6)). Based on the hypothesis that interaction between oxygen and radiation induced free-radicals is, at least in part, responsible for the biological damage, a specific water requirement for such damage could be expected.(5) CALDECOTT (4) a n d CURTIS (7) reported that the decreasing radiation damage at low to intermediate seed moisture was due to the recombination and neutralization of the freeradicals with increasing moisture levels in the presence of oxygen. The higher radiosensitivity at moisture levels beyond the point of minimum sensitivity has been attributed to the influence of increased metabolic activity of the seeds.(2,~a) The aim of the present work was to check this possibility mainly in terms of nucleic acid synthesis.
parameters, namely length of shoot and root, fresh and dry weights of shoot, root, scutellum, endosperm and seedlings. Seed moisture was determined after drying the specimens at 105°C for 48 hr. It was calculated as per cent of fi'esh-weight. The dry weight of different organs was determined after desiccation in an oven at 70°C. In another series, dry (3.4 per cent), moist (9"8 per cent) and wet (13.2 per cent moisture) seeds were irradiated with a total dose of 0 and 12.5 kR and allowed to germinate in the dark for 4 days at 25°C. Thymidine-6-aH (200 v.Ci/ nal, specific radioactivity 14 Ci/mM) was fed through cut endosperm for 6 hr to the duplicate groups of ten seedlings per treatment. The seedlings were thoroughly washed and divided into different organs; and their nucleic acid, t3rotein contents and radioactivity were measured. The protein content was determined in the fractions by the method of LOWRY et al.( TM The nucleic acids were estimated by the method of SCHMIDT-THANHAUSER (15) with the following modifications: alkali hydrolysis was performed for 24 hr at 37°C instead of 1 hr. Deproteinization was affected by chloroform, which improved the U.V. spectrum. RESULTS
MATERIALS AND METHODS
In order to obtain different seed moisture, Ingrid barley seeds were equilibrated over different concentrations of glycerol solutions (60, 70, 90 and 100 per cent) or dry calcium chloride, in desiccators at 25±1°C, following OSBORNE and BACON.(TM Seeds with different moisture content (3.4, 7.5, 9.8, 13.2 and 14-1 per cent) were simultaneously irradiated (embryo facing upwards) in a Maxitron X-ray machine at a dose rate of 1.25 kR/min 28.5 cm from the centre (300 kV, 20 mA, I m m Al) with a total dose of 0, I0"0 12"5 and 15"0 kR. Immediately after irradiation, the seeds were soaked in distilled water. Control and irradiated seeds (3 replications of 20 seeds per treatment) were grown at 25°C in Perlite, in the dark, for 3 days and then under artificial lighting (5,100 Lux) tbr 5 days. The radiation damage found in 8-day-old seedlings was estimated on the basis of different morphological
1. Physical and molphological observations Data in Table 1 show that the method used here was successful in producing different moisture content in the seeds. (a) Shoot and root length (Fig. 1). Increase in the radiation dose produced higher damage in both shoot and root. The relationship between radiation damage and moisture level was found to be inverse up to about 10 per cent seed moisture, and direct at higher moisture levels. (b) Fresh weight (Fig. 2). The fresh weight of shoot and root decreased with increase in the radiation dose. The relationship between the seed moisture and radiation damage was similar to that established by changes in organ length. The fresh weight of the irradiated endosperm was higher than that of controls. 'Moist' endosperms had the minimum fresh weight as compared to 'wet' and 'dry' ones. (c) Dry weight (Fig. 3). Both irradiation and moisture effected root, shoot and endosperm,
X-IRRADIATION AND SEED-MOISTURE ON METABOLISM IN BARLEY
439
Table 1. Moisture (as percentage of wet weight) in barley seeds afterfive weeks of storage at 25°C Moisture-stability chemical
Seed
Glycerol (by weight) Ingrid barley
60% 70% 90°,/o 100% Calcium chloride (anhydrous)
[ ] 3.4% Moisture I~1 "7.5% Moisture [ ] 9.8% Moisture []
13.2%Moisture
•
141% Moisture
Shoot
£
I0
Root
Control
I00 '
-..
12.5
x-radiation dose,
15.0
1 13-93 13.40 9.72 7.65 3.33
Moisture-content determination 2 3 Average 14.32 13"27 9.86 7.27 3.58
14-05 13.02 9.68 7.67 3.28
14.1 13-2 9.8 7.5 3.4
(b) Scutellum (Fig. 5). No effect of radiation nor of seed moisture could be detected on the AS, DNA, R N A or protein contents. Surprisingly, a significant increase in the specific r a d i o activity of the AS was observed in thymidine-3H treated plants, while the level of DNA remained unaffected. (c) Endosperm (Fig. 6). Compared to controls, AS, RNA, D N A and protein contents appeared to be higher in irradiated plants. No effect of seed moisture could be detected. (d) Root (Fig. 7). Radiation as well as moisture content had no effect on any of the parameters examined. (e) Whole plant (Fig. 8). Taking the plant as a whole, radiation or seed moisture appears to have no influence on RNA, DNA and protein content. However, AS appears to be dependent on moisture ; and radiation significantly depressed AS content.
kR
FzG. 1. Influence of X-irradiation and moisture content of barley seed, on shoot and root length. but not the scutellum. Interestingly, no significant effect was observed in the dry weight per plant.
2. Biochemical data (a) Shoot (Fig. 4). In non-irradiated plants, the seed moisture had no effect on AS, RNA, D N A and protein contents. In irradiated plants, the same were depressed by irradiation; the inhibition being least in the moist seeds. In thymidine-3H treated plants, there was no significant influence of seed moisture or radiation dose on the specific radioactivities of AS and DNA.
DISCUSSION
Though dry weight, AS, R N A and D N A content per organ decrease in the irradiated plant, they increase correspondingly in the endosperm, so that most of these parameters remain unchanged in the plant considered as a whole (Table 2 A). This shows that there is no net increase in the content of whole barley germinating in the dark. It would appear that irradiation at the dose used did not effect the breakdown ofmacromolecules in the endo.sperm. It did, however, impair the utilization of product by the embryo and as a result the breakdown products accumulated in the endosperm. The shoot appears to be highly sensitive to X-rays as evidenced by the D N A content, which may be considered to represent the number of cells. A
R. K. J O S H I and L. L E D O U X
4-4.0
[] [] [] [] •
I00 80
~
60 40 20 0
250 hoot
6O
~' 200
. ~ 150
°f ++
Root
40 .z=
3.4%Moisture 7-5%Moisture 9-8%Moisture 15.2%Moisture 14.1%Moisture
~= ¢1
L~
20
I00
0
Scutellum
Endosperm
0
0
Control
I0"0
Control
15-0
12'5
x-rodiolion dose,
I0.0
12,5
15-0
kR
Fro. 2. Influence of X-irradiation and moisture content of barley seed, on fresh-weight of different organs.
20
[ ] 3-4%Moisture
0
°• +13-2%Moislu,'s +++*o+[ "]
:f
•
,ot
~£'°oI
°~'~
I~
:~NA
14'1% Moisture
[~
~
,-~
s.o~
~
~
~
~
"oo'
'°I.
o--
60
~
30
°++ ++
[ ] 9.8% Moisture [ ] 13.2 %Moisture
s+m~,,om Prolem
i ! Acid-solube
~' 40
SO0
+;:I
200 Control
I0-0 x-tociiotion dose,
125 kR
15.0
FIG. 3. Influence of X-irradiation and moisture content of barley seed, on dry-weight of different organs.
I00 I
COntrol
12.5kR
ONA Control
12"5kR
FIo. 4. Influence of X-irradiation and moisture content of barley seed, on nucleic acid synthesis in shoot.
X - I R R A D I A T I O N A N D S E E D - M O I S T U R E O N METABOLISM IN BARLEY Acid-soluble
[ ] :5.4% Moislure [ ] 9 8 %Moisture t Acid-soluble
[ ] 13.2%Moisture
~
[] 3-4% Moisture
~IIB
[] 9.8% Moisture
"10 F DNA
Acid-soluble
i-I ]3.2% Moisture 6°I'RNA
J DNA
°:t
~
441
:~
~o
40
g
'°f
u
Protein
400
?-00l- Protein DNA
Control
Control
12 5kR
12 5kR
2(3C
F1o. 5. Influence of X-irradiation and moisture content of barley seed, on nucleic acid synthesis in scute]lum.
'2FJ DNA
0
Control
Control
12 5kR
IZ.SkR
Fro.7. Influence of X-irradiation and moisture content of barley seed, on nucleic acid synthesis in root. t
Acid-soluble [ ] 3-4% Moisture [ ] 9 8 % Moisture Acid-so}uble
= DNA
[ ] 13.2% Mobture
120 [ ] 3"4% Moisture
RNA 90-
90 60
[ ] 9.8% Moisture
IO0
30 [ ] 132%Moisture
60-
0 RNA ::L
30-
2ofAcdsou
2oc 150 50
:::L
Acid-soluble
300
Ioc
._~ 2oo >
u
Z5
u k=
50
DNA
125kR
~" 60
IOO
m
Control
so
50 0
~
~
Control
12.SkR
FIG. 6. Influence of X-irradiation and moisture content of barley seed, on nucleic acid synthesis in endosperm.
°
~a.
30
m
:r
Control
12.5kR
o DNA
=
Control
N
12.SkR
FIG. 8. Influence of X-irradiation and moisture content of barley seed, on nucleic acid synthesis in seedling.
442
R . K . J O S H I and L. L E D O U X Table 2. Influence of X-irradiation and seed-moisture on length, dry matter and nucleic acids content in the different organs of 4-day-old barley
A. Effect of X-rays
Organ Shoot Root Scutellum Endosperm Total plant
Length
Dry wt
AS
RNA
DNA
~ ~ ----
.J, ~ 0 l 0
~, 0 0 i .1
~ ~t 0 I 0
J, 0 0 t 0
Specific radioactivity AS DNA
Protein .!. 0 0 J" 0
0 0 'T' 0 --
0 0 0 0 --
B. Effect of seed-moisture
Organ
Shoot Root Scutellum Endosperm 0 i ! q-
Denotes Denotes Denotes Denotes
Non-irradiated seedling Dry ProLength wt AS RNA DNA tein
h-radiated seedling Dry Length wt AS RNA DNA
Prorein
0 0
0 0
0 0
0 0
0 0
0 0
+ +
+ +
+ 0
+ 0
+ 0
+ +
---
0 0
+ 0
0 0
0 0
0 0
---
0 0
0 0
0 0
0
0
0 0
no significant change. significant increase at 0.05 P level. significant decrease at 0.05 P level. maximum radioresistance at intermediate moisture content.
similar reduction in growth, nucleic acid content a n d protein synthesis in 4 - d a y - o l d b a r l e y seedlings as a result of neutron i r r a d i a t i o n of seeds was observed by MIKAELSEN. (1~) HUYSTEE a n d CHERRY, (9) on the other hand, found that while seedling growth was inhibited in p e a n u t seeds sown i m m e d i a t e l y after X - i r r a d i a t i o n , the nucleic acids were affected only after 4 weeks of post-irradiation storage. T h e possibility that translocation of macromolecules could be an i m p o r t a n t p h e n o m e n o n in this case has been discussed elsewhere.O0) W e can only point out here that the present results are not in disagreem e n t with this view considering the little effect of i r r a d i a t i o n on the specific r a d i o a c t i v i t y o f DNA. T a b l e 2 B summarizes the effect of v a r y i n g the moisture content o f the seed at the time o f irradiation. W h i l e no effect o f moisture could be recognized in n o n - i r r a d i a t e d seed, the a c t u a l moisture content a p p e a r e d to be o f g r e a t
i m p o r t a n c e when seeds were i r r a d i a t e d . NOTAN! a n d GAUR (13) a n d BmBL a n d MOSTAFA(1) have r e p o r t e d an i n t e r m e d i a t e level of moisture giving m a x i m u m radioresistance to seeds. O u r results confirm this observation. H o w e v e r , the variations in exact seed moisture contents giving similar effects, as observed by different workers, is p r o b a b l y d u e to different e x p e r i m e n t a l conditions a n d seed v a r i e t y e m p l o y e d . Also, it a p p e a r s here that such an effect is m o r e pron o u n c e d in shoot t h a n in root, scutellum or endosperm. I n shoots all p a r a m e t e r s m e a s u r e d (length, d r y weight, AS, R N A or D N A contents) were similarly affected. I n roots, length a n d d r y weight were affected b u t none of the o t h e r parameters. EHRENBERGand FALUDI-DANIEL(s)studied the changes in various fractions o f b a r l e y e m b r y o after y - i r r a d i a t i o n o f seeds; they observed a r e d u c t i o n in a~p i n c o r p o r a t i o n in free nucleotide pool as a result o f r a d i a t i o n and, also, decrease
X - I R R A D I A T I O N AND S E E D - M O I S T U R E ON M E T A B O L I S M IN BARLEY in seed moisture content. T h e i r studies also showed no difference in the i n c o r p o r a t i o n into R N A a n d D N A relative to seed moisture, or r a d i a t i o n . D a t a from the t h y m i d i n e - S H studies r e p o r t e d here a r e in a g r e e m e n t with these observations. I t a p p e a r s therefore unlikely t h a t the changes in radiosensitivity observed a t different moisture levels are due to w a t e r - i n d u c e d modifications o f m e t a b o l i c activities. I t is, however, possible t h a t the moisture c o n t e n t of the seed m i g h t affect the a v a i l a b i l i t y of the m a c r o m o l e c u l e s necessary for the g e r m i n a t i o n of barley, in the absence o f photosynthesis.
Acknowledgements--This work was carried out during the tenure of a scholarship from Ministers des Affaires Etrangeres et de commerce Exterieur (O.C.D.) to one of the authors (R.K.J.). The authors also thank Dr. J. R. MAISIN,Director, Department of Radiobiology, (C.E.N.) and Dr. A. R. GOPAL-AYENGAR,Director, Bio-Medical Group, Bhabha Atomic Research Centre, Bombay, for their keen interest and encouragement.
REFERENCES 1. BIEBL R. and MOSTAFA I. Y. (1965) Water content of wheat and barley seeds and their radiosensitivity. Radiation Botany 5, 1-6. 2. BmBL R. and PAPER. (1951) R6ntgenstrahlen Wirkungen auf Keimenden Weizen. Oesterr. Botan. Z. 98, 361-382. 3. CALDECOTT R. S. (1954) Inverse relationship between the water content of seeds and their sensitivity to X-rays. Science 120, 809-810. 4. CALDECOTT R. S. (1955) Reduction in X-ray sensitivity of seeds by hydration. Nature 176, 306. 5. CONaER A. D. (1961) Biological after-effect and long-lived radicals in irradiated seeds. 07. Cell. Comp. Physiol. (Suppl. I) 511, 27-32.
443
6. CoNosg B. V., NILAN R. A. and KONZAK C. F. (1968) Post irradiation oxygen sensitivity of barley seeds varying slightly in water content. Radiation Botany 8, 31-36. 7. CURTIS H . J . (I961) Delayed effects of radiation on seeds, pp. 193-204. In P. L. T. ILBERRY (ed.), Radiobiology. Butterworths, London. 8. F,HRENBERG L. and FALLUDI-DANIELA. (1967) Phosphorous metabolism of barley embryos in relation to radiation damage. Radiation Botany 7, 409-414. 9. HUYSTEE R. V. and CHERRY J. H. (1967) Effect of X-irradiation and post-irradiation storage of peanut seed on nucleic acid metabolism in cotyledons. Radiation Botany 7, 217-223. 10. LEDOUX L. (1962) Effect of X-rays on the tramlocation of labelled nucleic-acids and proteins in germinating barley, pp. 175-182. In, Biological
effects of ionizhzg radiation at the molecular level. IAEA, Vienna. 11. LOWRY 0 . H., ROSENBROUGHN. J., LEwxs F. A. and R~qDALL R . J . (1951) Protein measurement with the Folin Phenol reagent. J. Biol. Chem. 193, 265-275. 12. MX~LSEN K. (1967) Effects of fast neutrons on seedling growth and metabolism in barley, pp. 63-70. In, Neutron irradiation of seeds II. FAO[ IAEA Teeh. Rep. 92. IAEA, Vienna. 13. NOTANIN. K. and GAURB. K. (1962) Paradoxical modifications in radiosensitivity of maize and barley seeds stabilized for different moisture contents, pp. 443--453. In, Biological effects of ionizing radiation at the molecular level. IAEA, Vienna. 14. OSBORNET. S. and BACON-JAM~SA. (1961) Two improved and inexpensive systems for moisture stabilization in seeds or other tissues. Plant Physiol. 36, 309-312. 15. SCHMIDT G. and THANHAUSER S. J. (1945) A method for the determination of deoxyribonucleicacid and phosphoproteins in animal tissues. 07. Biol. Chem. 161, 83-89.
INFLUENCE OF X - I R R A D I A T I O N AND SEED-MOISTURE ON NUCLEIC-ACID AND P R O T E I N METABOLISM IN BARLEY* R. K. JOSHI Biology Division, Bhabha Atomic Research Centre, Trombay, Bombay-85, India ,*rid L. L E D O U X Biochimie Cellulaire, D~partment de Radiobiologie, Centre d'Etude de l'Energie Nucl~aire, Mol, Belgium
(Received 6 February 1970) Abstract---Seeds with varying moisture content (3-14 per cent) were X-irradiated with doses of 0, 10, 12-5 and 15 kR. Radiation-induced changes in the length of root and shoot, and their fresh and dry weights indicate that the seeds show maximum radioresistance at intermediate moisture levels and that the shoot is more sensitive to X-rays than the root. There was no significant modification of DNA, RNA and acid soluble (AS) content while protein content was adversely affected by radiation irrespective of seed moisture. However, no change in total macromolecular content could be detected in the plant as a whole. R~smm~----Des graines poss~dant des teneurs en eau vari~es (3 ~ 14%) ont ~tfi irradi~es par les rayons X aux doses de 0, 10, 12,5 et 15 kR. Les changements de longueur des racines et des apex induits par les radiations, ainsi que les poids frais et secs indiquent que les graines montrent un maximum de radio-r~sistance ~t des niveaux d'hydratation interm~diaires et que l'apex est plus sensible que la racine. I1 n'y a pas de modification significative des contenus en DNA, R N A ainsi qu'en acido-soluble alors que le contenu en protdines est invers~ment influenc~ par les rayons ind~pendamment de l'hydratation. On n'a cependant pu d~tecter de changement en contenu total des macromolficules des plantes enti6res. Z u s a m m e n f a s s u n g - - S a m e n mit verschiedenem Gehalt an Feuchtigkeit (3 bis 14%) wurden mit R~ntgenstrahlen yon 0, 10, 12,5 und 15 kR behandelt. Strahleninduzierte Ver~inderungen der L/inge der Wurzeln und Sch6sslinge und ihr Frisch- und Trockengewicht zeigen, dass die Samen den h6chsten Grad an Strahlenresistenz bei mittlerem Feuchtigkeitsgehalt aufweisen, und dass die Sch6sslinge gegeniiber den R6ntgenstrahlen empfindlicher sind als die Wurzeln. Es trat keine signifikante Modifikation der DNS, RNS und des s~iurel6slichen Gehaltes auf, dagegen wurde der Proteingehalt v o n d e r Strahlung beeinflusst ungeaehtet der Feuchtigkeit der Samen. Hinsichtlich des gesamten makromolikularen Gehaltes konnte aber in der Pflanze als ganze keine Ver~inderung festgestellt werden.
* This work was carried out at Section de Biochimie Cellulaire, D6partment de Radiobiologie, Centre d'Etude de l'Energie Nucl6aire, Mol, Belgium. 437
R. K. JOSItI and L. LEDOUX
438 INTRODUCTION
THE EFFECTS of sparsely ionizing radiations on seeds, in relation to their moisture content have been extensively studied (CALD~.COT'r,(3,4) BmBL and PAP~.(2)). The radiosensitivity of seeds varies greatly even with a little change in seed moisture (CoNOER et al.(6)). According to NOTANI and GAUR03) it appears to be minimal at intermediate moisture content and enhanced above and below this range. Such a relationship is oxygen-dependent (CoNOER el al.(6)). Based on the hypothesis that interaction between oxygen and radiation induced free-radicals is, at least in part, responsible for the biological damage, a specific water requirement for such damage could be expected.(5) CALDECOTT (4) a n d CURTIS (7) reported that the decreasing radiation damage at low to intermediate seed moisture was due to the recombination and neutralization of the freeradicals with increasing moisture levels in the presence of oxygen. The higher radiosensitivity at moisture levels beyond the point of minimum sensitivity has been attributed to the influence of increased metabolic activity of the seeds.(2,~a) The aim of the present work was to check this possibility mainly in terms of nucleic acid synthesis.
parameters, namely length of shoot and root, fresh and dry weights of shoot, root, scutellum, endosperm and seedlings. Seed moisture was determined after drying the specimens at 105°C for 48 hr. It was calculated as per cent of fi'esh-weight. The dry weight of different organs was determined after desiccation in an oven at 70°C. In another series, dry (3.4 per cent), moist (9"8 per cent) and wet (13.2 per cent moisture) seeds were irradiated with a total dose of 0 and 12.5 kR and allowed to germinate in the dark for 4 days at 25°C. Thymidine-6-aH (200 v.Ci/ nal, specific radioactivity 14 Ci/mM) was fed through cut endosperm for 6 hr to the duplicate groups of ten seedlings per treatment. The seedlings were thoroughly washed and divided into different organs; and their nucleic acid, t3rotein contents and radioactivity were measured. The protein content was determined in the fractions by the method of LOWRY et al.( TM The nucleic acids were estimated by the method of SCHMIDT-THANHAUSER (15) with the following modifications: alkali hydrolysis was performed for 24 hr at 37°C instead of 1 hr. Deproteinization was affected by chloroform, which improved the U.V. spectrum. RESULTS
MATERIALS AND METHODS
In order to obtain different seed moisture, Ingrid barley seeds were equilibrated over different concentrations of glycerol solutions (60, 70, 90 and 100 per cent) or dry calcium chloride, in desiccators at 25±1°C, following OSBORNE and BACON.(TM Seeds with different moisture content (3.4, 7.5, 9.8, 13.2 and 14-1 per cent) were simultaneously irradiated (embryo facing upwards) in a Maxitron X-ray machine at a dose rate of 1.25 kR/min 28.5 cm from the centre (300 kV, 20 mA, I m m Al) with a total dose of 0, I0"0 12"5 and 15"0 kR. Immediately after irradiation, the seeds were soaked in distilled water. Control and irradiated seeds (3 replications of 20 seeds per treatment) were grown at 25°C in Perlite, in the dark, for 3 days and then under artificial lighting (5,100 Lux) tbr 5 days. The radiation damage found in 8-day-old seedlings was estimated on the basis of different morphological
1. Physical and molphological observations Data in Table 1 show that the method used here was successful in producing different moisture content in the seeds. (a) Shoot and root length (Fig. 1). Increase in the radiation dose produced higher damage in both shoot and root. The relationship between radiation damage and moisture level was found to be inverse up to about 10 per cent seed moisture, and direct at higher moisture levels. (b) Fresh weight (Fig. 2). The fresh weight of shoot and root decreased with increase in the radiation dose. The relationship between the seed moisture and radiation damage was similar to that established by changes in organ length. The fresh weight of the irradiated endosperm was higher than that of controls. 'Moist' endosperms had the minimum fresh weight as compared to 'wet' and 'dry' ones. (c) Dry weight (Fig. 3). Both irradiation and moisture effected root, shoot and endosperm,
X-IRRADIATION AND SEED-MOISTURE ON METABOLISM IN BARLEY
439
Table 1. Moisture (as percentage of wet weight) in barley seeds afterfive weeks of storage at 25°C Moisture-stability chemical
Seed
Glycerol (by weight) Ingrid barley
60% 70% 90°,/o 100% Calcium chloride (anhydrous)
[ ] 3.4% Moisture I~1 "7.5% Moisture [ ] 9.8% Moisture []
13.2%Moisture
•
141% Moisture
Shoot
£
I0
Root
Control
I00 '
-..
12.5
x-radiation dose,
15.0
1 13-93 13.40 9.72 7.65 3.33
Moisture-content determination 2 3 Average 14.32 13"27 9.86 7.27 3.58
14-05 13.02 9.68 7.67 3.28
14.1 13-2 9.8 7.5 3.4
(b) Scutellum (Fig. 5). No effect of radiation nor of seed moisture could be detected on the AS, DNA, R N A or protein contents. Surprisingly, a significant increase in the specific r a d i o activity of the AS was observed in thymidine-3H treated plants, while the level of DNA remained unaffected. (c) Endosperm (Fig. 6). Compared to controls, AS, RNA, D N A and protein contents appeared to be higher in irradiated plants. No effect of seed moisture could be detected. (d) Root (Fig. 7). Radiation as well as moisture content had no effect on any of the parameters examined. (e) Whole plant (Fig. 8). Taking the plant as a whole, radiation or seed moisture appears to have no influence on RNA, DNA and protein content. However, AS appears to be dependent on moisture ; and radiation significantly depressed AS content.
kR
FzG. 1. Influence of X-irradiation and moisture content of barley seed, on shoot and root length. but not the scutellum. Interestingly, no significant effect was observed in the dry weight per plant.
2. Biochemical data (a) Shoot (Fig. 4). In non-irradiated plants, the seed moisture had no effect on AS, RNA, D N A and protein contents. In irradiated plants, the same were depressed by irradiation; the inhibition being least in the moist seeds. In thymidine-3H treated plants, there was no significant influence of seed moisture or radiation dose on the specific radioactivities of AS and DNA.
DISCUSSION
Though dry weight, AS, R N A and D N A content per organ decrease in the irradiated plant, they increase correspondingly in the endosperm, so that most of these parameters remain unchanged in the plant considered as a whole (Table 2 A). This shows that there is no net increase in the content of whole barley germinating in the dark. It would appear that irradiation at the dose used did not effect the breakdown ofmacromolecules in the endo.sperm. It did, however, impair the utilization of product by the embryo and as a result the breakdown products accumulated in the endosperm. The shoot appears to be highly sensitive to X-rays as evidenced by the D N A content, which may be considered to represent the number of cells. A
R. K. J O S H I and L. L E D O U X
4-4.0
[] [] [] [] •
I00 80
~
60 40 20 0
250 hoot
6O
~' 200
. ~ 150
°f ++
Root
40 .z=
3.4%Moisture 7-5%Moisture 9-8%Moisture 15.2%Moisture 14.1%Moisture
~= ¢1
L~
20
I00
0
Scutellum
Endosperm
0
0
Control
I0"0
Control
15-0
12'5
x-rodiolion dose,
I0.0
12,5
15-0
kR
Fro. 2. Influence of X-irradiation and moisture content of barley seed, on fresh-weight of different organs.
20
[ ] 3-4%Moisture
0
°• +13-2%Moislu,'s +++*o+[ "]
:f
•
,ot
~£'°oI
°~'~
I~
:~NA
14'1% Moisture
[~
~
,-~
s.o~
~
~
~
~
"oo'
'°I.
o--
60
~
30
°++ ++
[ ] 9.8% Moisture [ ] 13.2 %Moisture
s+m~,,om Prolem
i ! Acid-solube
~' 40
SO0
+;:I
200 Control
I0-0 x-tociiotion dose,
125 kR
15.0
FIG. 3. Influence of X-irradiation and moisture content of barley seed, on dry-weight of different organs.
I00 I
COntrol
12.5kR
ONA Control
12"5kR
FIo. 4. Influence of X-irradiation and moisture content of barley seed, on nucleic acid synthesis in shoot.
X - I R R A D I A T I O N A N D S E E D - M O I S T U R E O N METABOLISM IN BARLEY Acid-soluble
[ ] :5.4% Moislure [ ] 9 8 %Moisture t Acid-soluble
[ ] 13.2%Moisture
~
[] 3-4% Moisture
~IIB
[] 9.8% Moisture
"10 F DNA
Acid-soluble
i-I ]3.2% Moisture 6°I'RNA
J DNA
°:t
~
441
:~
~o
40
g
'°f
u
Protein
400
?-00l- Protein DNA
Control
Control
12 5kR
12 5kR
2(3C
F1o. 5. Influence of X-irradiation and moisture content of barley seed, on nucleic acid synthesis in scute]lum.
'2FJ DNA
0
Control
Control
12 5kR
IZ.SkR
Fro.7. Influence of X-irradiation and moisture content of barley seed, on nucleic acid synthesis in root. t
Acid-soluble [ ] 3-4% Moisture [ ] 9 8 % Moisture Acid-so}uble
= DNA
[ ] 13.2% Mobture
120 [ ] 3"4% Moisture
RNA 90-
90 60
[ ] 9.8% Moisture
IO0
30 [ ] 132%Moisture
60-
0 RNA ::L
30-
2ofAcdsou
2oc 150 50
:::L
Acid-soluble
300
Ioc
._~ 2oo >
u
Z5
u k=
50
DNA
125kR
~" 60
IOO
m
Control
so
50 0
~
~
Control
12.SkR
FIG. 6. Influence of X-irradiation and moisture content of barley seed, on nucleic acid synthesis in endosperm.
°
~a.
30
m
:r
Control
12.5kR
o DNA
=
Control
N
12.SkR
FIG. 8. Influence of X-irradiation and moisture content of barley seed, on nucleic acid synthesis in seedling.
442
R . K . J O S H I and L. L E D O U X Table 2. Influence of X-irradiation and seed-moisture on length, dry matter and nucleic acids content in the different organs of 4-day-old barley
A. Effect of X-rays
Organ Shoot Root Scutellum Endosperm Total plant
Length
Dry wt
AS
RNA
DNA
~ ~ ----
.J, ~ 0 l 0
~, 0 0 i .1
~ ~t 0 I 0
J, 0 0 t 0
Specific radioactivity AS DNA
Protein .!. 0 0 J" 0
0 0 'T' 0 --
0 0 0 0 --
B. Effect of seed-moisture
Organ
Shoot Root Scutellum Endosperm 0 i ! q-
Denotes Denotes Denotes Denotes
Non-irradiated seedling Dry ProLength wt AS RNA DNA tein
h-radiated seedling Dry Length wt AS RNA DNA
Prorein
0 0
0 0
0 0
0 0
0 0
0 0
+ +
+ +
+ 0
+ 0
+ 0
+ +
---
0 0
+ 0
0 0
0 0
0 0
---
0 0
0 0
0 0
0
0
0 0
no significant change. significant increase at 0.05 P level. significant decrease at 0.05 P level. maximum radioresistance at intermediate moisture content.
similar reduction in growth, nucleic acid content a n d protein synthesis in 4 - d a y - o l d b a r l e y seedlings as a result of neutron i r r a d i a t i o n of seeds was observed by MIKAELSEN. (1~) HUYSTEE a n d CHERRY, (9) on the other hand, found that while seedling growth was inhibited in p e a n u t seeds sown i m m e d i a t e l y after X - i r r a d i a t i o n , the nucleic acids were affected only after 4 weeks of post-irradiation storage. T h e possibility that translocation of macromolecules could be an i m p o r t a n t p h e n o m e n o n in this case has been discussed elsewhere.O0) W e can only point out here that the present results are not in disagreem e n t with this view considering the little effect of i r r a d i a t i o n on the specific r a d i o a c t i v i t y o f DNA. T a b l e 2 B summarizes the effect of v a r y i n g the moisture content o f the seed at the time o f irradiation. W h i l e no effect o f moisture could be recognized in n o n - i r r a d i a t e d seed, the a c t u a l moisture content a p p e a r e d to be o f g r e a t
i m p o r t a n c e when seeds were i r r a d i a t e d . NOTAN! a n d GAUR (13) a n d BmBL a n d MOSTAFA(1) have r e p o r t e d an i n t e r m e d i a t e level of moisture giving m a x i m u m radioresistance to seeds. O u r results confirm this observation. H o w e v e r , the variations in exact seed moisture contents giving similar effects, as observed by different workers, is p r o b a b l y d u e to different e x p e r i m e n t a l conditions a n d seed v a r i e t y e m p l o y e d . Also, it a p p e a r s here that such an effect is m o r e pron o u n c e d in shoot t h a n in root, scutellum or endosperm. I n shoots all p a r a m e t e r s m e a s u r e d (length, d r y weight, AS, R N A or D N A contents) were similarly affected. I n roots, length a n d d r y weight were affected b u t none of the o t h e r parameters. EHRENBERGand FALUDI-DANIEL(s)studied the changes in various fractions o f b a r l e y e m b r y o after y - i r r a d i a t i o n o f seeds; they observed a r e d u c t i o n in a~p i n c o r p o r a t i o n in free nucleotide pool as a result o f r a d i a t i o n and, also, decrease
X - I R R A D I A T I O N AND S E E D - M O I S T U R E ON M E T A B O L I S M IN BARLEY in seed moisture content. T h e i r studies also showed no difference in the i n c o r p o r a t i o n into R N A a n d D N A relative to seed moisture, or r a d i a t i o n . D a t a from the t h y m i d i n e - S H studies r e p o r t e d here a r e in a g r e e m e n t with these observations. I t a p p e a r s therefore unlikely t h a t the changes in radiosensitivity observed a t different moisture levels are due to w a t e r - i n d u c e d modifications o f m e t a b o l i c activities. I t is, however, possible t h a t the moisture c o n t e n t of the seed m i g h t affect the a v a i l a b i l i t y of the m a c r o m o l e c u l e s necessary for the g e r m i n a t i o n of barley, in the absence o f photosynthesis.
Acknowledgements--This work was carried out during the tenure of a scholarship from Ministers des Affaires Etrangeres et de commerce Exterieur (O.C.D.) to one of the authors (R.K.J.). The authors also thank Dr. J. R. MAISIN,Director, Department of Radiobiology, (C.E.N.) and Dr. A. R. GOPAL-AYENGAR,Director, Bio-Medical Group, Bhabha Atomic Research Centre, Bombay, for their keen interest and encouragement.
REFERENCES 1. BIEBL R. and MOSTAFA I. Y. (1965) Water content of wheat and barley seeds and their radiosensitivity. Radiation Botany 5, 1-6. 2. BmBL R. and PAPER. (1951) R6ntgenstrahlen Wirkungen auf Keimenden Weizen. Oesterr. Botan. Z. 98, 361-382. 3. CALDECOTT R. S. (1954) Inverse relationship between the water content of seeds and their sensitivity to X-rays. Science 120, 809-810. 4. CALDECOTT R. S. (1955) Reduction in X-ray sensitivity of seeds by hydration. Nature 176, 306. 5. CONaER A. D. (1961) Biological after-effect and long-lived radicals in irradiated seeds. 07. Cell. Comp. Physiol. (Suppl. I) 511, 27-32.
443
6. CoNosg B. V., NILAN R. A. and KONZAK C. F. (1968) Post irradiation oxygen sensitivity of barley seeds varying slightly in water content. Radiation Botany 8, 31-36. 7. CURTIS H . J . (I961) Delayed effects of radiation on seeds, pp. 193-204. In P. L. T. ILBERRY (ed.), Radiobiology. Butterworths, London. 8. F,HRENBERG L. and FALLUDI-DANIELA. (1967) Phosphorous metabolism of barley embryos in relation to radiation damage. Radiation Botany 7, 409-414. 9. HUYSTEE R. V. and CHERRY J. H. (1967) Effect of X-irradiation and post-irradiation storage of peanut seed on nucleic acid metabolism in cotyledons. Radiation Botany 7, 217-223. 10. LEDOUX L. (1962) Effect of X-rays on the tramlocation of labelled nucleic-acids and proteins in germinating barley, pp. 175-182. In, Biological
effects of ionizhzg radiation at the molecular level. IAEA, Vienna. 11. LOWRY 0 . H., ROSENBROUGHN. J., LEwxs F. A. and R~qDALL R . J . (1951) Protein measurement with the Folin Phenol reagent. J. Biol. Chem. 193, 265-275. 12. MX~LSEN K. (1967) Effects of fast neutrons on seedling growth and metabolism in barley, pp. 63-70. In, Neutron irradiation of seeds II. FAO[ IAEA Teeh. Rep. 92. IAEA, Vienna. 13. NOTANIN. K. and GAURB. K. (1962) Paradoxical modifications in radiosensitivity of maize and barley seeds stabilized for different moisture contents, pp. 443--453. In, Biological effects of ionizing radiation at the molecular level. IAEA, Vienna. 14. OSBORNET. S. and BACON-JAM~SA. (1961) Two improved and inexpensive systems for moisture stabilization in seeds or other tissues. Plant Physiol. 36, 309-312. 15. SCHMIDT G. and THANHAUSER S. J. (1945) A method for the determination of deoxyribonucleicacid and phosphoproteins in animal tissues. 07. Biol. Chem. 161, 83-89.