Planta (Berl.) 124, 303--309 (1975) 9 by Springer-Verlag 1975
Studies of Seed Development in Pisum sativum I I . R i b o s o m a l R N A C o n t e n t s in R e c i p r o c a l Crosses D. R o y Davies a n d V e r i t y B r e w s t e r John Innes Institute, Colney Lane, Norwich NR 4 7 UH, U. K. Received 13 March; accepted 2 April 1975
Summary. Comparisons have been made at different developmental stages of the amounts of ribosomal P~NA (rRNA) and of DNA in the cotyledons of two varieties of peas having different seed sizes. That with large seed has more rRNA per cell. The nature of the control of rRNA has been examined by the use of reciprocal crosses between the varieties, rRNA contents in the cells of a seed are not primarily dependent on the amounts of rDNA in those cells, but are regulated by the maternal parent; two hypotheses of the nature of this control are considered. Introduction The size of t h e seed in a n y given v a r i e t y is a function n o t o n l y of t h e a m o u n t s of p h o t o s y n t h a t e t h a t are available to be t r a n s l o e a t e d to t h a t sink a n d of t h e efficiency of t r a n s l o c a t i o n ; it is also d e p e n d e n t on t h e h o r m o n a l s t a t u s of t h e p l a n t a n d also on certain p a r a m e t e r s which are a t least in p a r t a u t o n o m o u s l y r e g u l a t e d w i t h i n t h e seed itself. These include t h e n u m b e r s of cells w i t h i n t h e seed a n d t h e weight of t h e cells (Davies, 1975). I n t u r n these are likely to be r e l a t e d to D N A a n d R N A contents, enzymic activities a n d R N A activities of t h e cells of t h e seed. W e are a t t e m p t i n g to e v a l u a t e b o t h t h e e x t e n t of t h e v a r i a t i o n in these p a r a m e t e r s in seed of different varieties of Pisum sativum, a n d also t h e n a t u r e of t h e control of these p a r a m e t e r s in seed d e v e l o p m e n t . I t is likely t h a t t h e r e g u l a t i o n of t h e p a r a m e t e r s described is a function b o t h of t h e seed's own g e n o t y p e a n d information c o n t e n t (intrinsic control) a n d also of t h e m a t e r n a l e n v i r o n m e n t of t h e p l a n t (extrinsic control). A m e t h o d of helping to d i s c r i m i n a t e b e t w e e n t h e r e l a t i v e roles of these two r e g u l a t o r y systems involves t h e e x p l o i t a t i o n of reciprocal crosses. I n a previous p a p e r (Davies, 1975) such crosses were used to e x a m i n e t h e role of cell n u m b e r , d r y a n d wet cell weight, g r o w t h rate, w a t e r c o n t e n t a n d g r o w t h period as d e t e r m i n a n t s of seed size a n d to e x a m i n e how t h e y are regulated. I n this r e p o r t studies of t h e nuclear D N A a n d of t h e r i b o s o m a l R N A (rRNA) contents of t h e c o t y l e d o n cells are described a n d t h e results considered in r e l a t i o n to t h e two s y s t e m s of control.
Materials and Methods Two varieties of Pisum sativum and their reciprocal hybrids were used; J I 430 (cv Greenshaft) and J I 181 (Keeran Pea from Nepal). The mean dry seed weights (in g) of J I 430,430 • 181,181 and 181 • 430 are 0.244:, 0.246, 0.073 and 0.111 respectively. DNA Contents. Tissues fixed in 3 parts ethanol: 1 part glacial acetic acid were hydrolysed in 5N HC1 at 20~ for 60 min, washed three times with distilled water and stained with Feulgen for 60 rain. They were then rinsed in fresh SO2 water and mounted on a coverslip in glycerol and covered with cellophane. This allowed the use of a crushing condenser to flatten the nuclei of the ee]ls prior to measuring their DNA content in a Vickers M85 microdensitometer. DNA values from at least 100 cells per sample were measured.
304
D. 1~. Davies and V. Brewster
Ribosomal RNA Contents. The amount of RNA in the cotyledons of J I 181 and J I 430 and their reciprocal hybrids was examined at different developmental stages and the proportions of different I~NAs determined in J I 181 and J I 430. To determine the proportions of 25s, 18s, 5s and 4s RNA at different stages, the I~NA was extracted by the method of Weinmann (1972) with the exception that 0,5% and 1.0% Sodium •-lauroyl sarcosinate was used as the detergent in their solutions I and I I I respectively. If double gels, 10% and 2,5% acrylamide, (Loening, 1967) were used for separating the I~NA species, it was impossible to accurately resolve the amount of 5s and 4s present without grossly overloading the gels for the other species of I~NA. Separate 2.5 % and 10 % gels were therefore used which could be loaded accordingly. Gels were run at 5 mA/gel at 4~ using Weinmann's (1972) buffer, after prerunning for 45 min. The gels were scanned in a Joyee-Locbl Chromoscan and the proportions of the different l ~ A s detected. Total RNA per cotyledon was routinely extracted either by the method of Click and Hackett (1966), and estimated by the orcinol method (Schneider, 1957) or by a modification of the method of Wilcoekson (1973) and estimated at OD 260. The estimates of RNA contents were identical with the two methods.
Results D N A Contents
Since t h e r e is a correlation b e t w e e n cell size a n d D N A c o n t e n t (Bennett, 1972) t h e nuclear D N A contents of t h e cotyledons of t h e varieties J I 181 a n d 430 were compared. These varieties have s u b s t a n t i a l different seed a n d cell weights (Davies, 1975). The results of t h e D N A e s t i m a t i o n s are shown in Fig. 1 t o g e t h e r w i t h d a t a from a r o o t t i p m e r i s t e m of J I 181. The l a t t e r has peaks representing the 2C a n d 4C values of G1 a n d G 2 cells r e s p e c t i v e l y A t 18 a n d 21 days, t h e cells of J I 181 h a d increased t h e i r D N A contents more r a p i d l y t h a n those of J I 430 b u t t h e r e was no clear difference b e t w e e n t h e two g e n o t y p e s thereafter. B o t h showed a wide s p r e a d of values u p to 64C, a n d a range of cell volumes was also observed. T h e a p p a r e n t r e d u c t i o n a t l a t e r stages (33 a n d 36 days) a l m o s t c e r t a i n l y is an a r t i f a c t arising from t h e difficulty of a d e q u a t e l y squashing t h e nuclei of t h e m o r e m a t u r e cells with t h e i r dense cellular contents. There is no evidence in these d a t a t h a t differences in t h e average cell weight of J I 181 a n d J I 430 can be a t t r i b u t e d to gross differences in D N A contents t h o u g h t h e resolution of t h e m e a s u r e m e n t s is such t h a t a slightly higher p r o p o r t i o n of larger cells a n d nuclei in J I 430 would n o t h a v e been detected. To d e t e c t this it would be necessary to s a m p l e s u b s t a n t i a l l y m o r e cells t h a n t h e ~ 100 scored in t h e p r e s e n t experiments. Ribosomal R N A Contents
The p r o p o r t i o n of 25s, 18s, 5s a n d 4s R N A d i d n o t differ b e t w e e n t h e g e n o t y p e s J I 181 a n d J I 430 or between t h e different stages e x a m i n e d from 18 d a y s to 27 d a y s after flowering. 25s plus 18s R N A c o n s t i t u t e d a p p r o x i m a t e l y 89 % of t h e t o t a l I~NA in J I 181 a n d 85% in J I 4 3 0 (Fig. 2). I n view of this, s u b s e q u e n t results are q u o t e d in t e r m s of t o t a l R N A a m o u n t s . The results for t h e R N A a m o u n t s are given in Fig. 3, p l o t t i n g t h e values a g a i n s t weights of c o t y l e d o n r a t h e r t h a n d a y s ; t h e p a t t e r n of results is t h e same with either b u t in c o m p a r i n g seed from different plants, t i m e is often a less a c c u r a t e d e t e r m i n a n t of d e v e l o p m e n t a l stage t h a n weight of cotyledon. A n e x p a n d e d scale is used for t h e J I 181 a n d J I 181 • 430 d a t a to p r e v e n t clustering of points, b u t t h e regression values shown, indicate t h a t
Studies of Seed Development in Pisum sativum
305
JI 181 JI 430
24 I
,
L
i
27 P
30
33qtoM;-, 4
6
8
np, ~
,
10
4
6
8
1]],
10
DNA/cell {log 2)
Fig. 1. DNA amounts (in arbitrary units) per cotyledon cell of JI 181 and JI 430, RT = root tips; 18 to 36 refers to developmental stage in number of days after flowering
the slopes of the lines are similar for all genotypes. The t~NA per unit weight of fresh cotyledons is constant for all genotypes and developmental stages examined (Fig. 4) even though this unit weight is made up different numbers of cells in the different genotypes. Since no cell division is occurring in the period depicted from 18 days after flowering onwards, the increase in RNA represents a very marked increase per cell. I n J I 4 3 0 and J I 4 3 0 • 181 this is approximately 9 fold (over 15 days), but in J I 181 and J I 181 • 430 only approximately 3 fold,--all having similar initial values at 18 days. I t is here that we see a clear cut distinction between small and large seeded types. There is first a marked difference in the final average amount of t~NA per cell, J I 430 having nearly 2.5 times as much R N A
306
D. R. Davies and V. Brewster
25s
A
18s
F i g . 2 A and B. Chromoscan traces of R:NA extracted from J I 430. (a) 2.5% acrylamide gel showing 25s a n d 18s peaks; (b) 10% acrylamide gel showing 5s and 4s peaks. Five times as much R~NA was located on to the 10% as onto the 2.5% gel
//~
10 . c80
Z~ 7:
o
ooOO
0
~
6-
o
X
e-
< 3 z a: 2
20 100
40 60 200 300 wet wt.in mg/cotytedon
80 (181f181xZ,30) 400 (430f130x181)
Fig. 3. RNA per cotyledon a~ different developmental stages. J I 430 o; J I 430 x 181 []; J I 181 O; J I 181 x 430 9 .8'
~7 ~6 o
h x 3
I0
L
Studies of Seed Development in Pisum sativum I I . R i b o s o m a l R N A C o n t e n t s in R e c i p r o c a l Crosses D. R o y Davies a n d V e r i t y B r e w s t e r John Innes Institute, Colney Lane, Norwich NR 4 7 UH, U. K. Received 13 March; accepted 2 April 1975
Summary. Comparisons have been made at different developmental stages of the amounts of ribosomal P~NA (rRNA) and of DNA in the cotyledons of two varieties of peas having different seed sizes. That with large seed has more rRNA per cell. The nature of the control of rRNA has been examined by the use of reciprocal crosses between the varieties, rRNA contents in the cells of a seed are not primarily dependent on the amounts of rDNA in those cells, but are regulated by the maternal parent; two hypotheses of the nature of this control are considered. Introduction The size of t h e seed in a n y given v a r i e t y is a function n o t o n l y of t h e a m o u n t s of p h o t o s y n t h a t e t h a t are available to be t r a n s l o e a t e d to t h a t sink a n d of t h e efficiency of t r a n s l o c a t i o n ; it is also d e p e n d e n t on t h e h o r m o n a l s t a t u s of t h e p l a n t a n d also on certain p a r a m e t e r s which are a t least in p a r t a u t o n o m o u s l y r e g u l a t e d w i t h i n t h e seed itself. These include t h e n u m b e r s of cells w i t h i n t h e seed a n d t h e weight of t h e cells (Davies, 1975). I n t u r n these are likely to be r e l a t e d to D N A a n d R N A contents, enzymic activities a n d R N A activities of t h e cells of t h e seed. W e are a t t e m p t i n g to e v a l u a t e b o t h t h e e x t e n t of t h e v a r i a t i o n in these p a r a m e t e r s in seed of different varieties of Pisum sativum, a n d also t h e n a t u r e of t h e control of these p a r a m e t e r s in seed d e v e l o p m e n t . I t is likely t h a t t h e r e g u l a t i o n of t h e p a r a m e t e r s described is a function b o t h of t h e seed's own g e n o t y p e a n d information c o n t e n t (intrinsic control) a n d also of t h e m a t e r n a l e n v i r o n m e n t of t h e p l a n t (extrinsic control). A m e t h o d of helping to d i s c r i m i n a t e b e t w e e n t h e r e l a t i v e roles of these two r e g u l a t o r y systems involves t h e e x p l o i t a t i o n of reciprocal crosses. I n a previous p a p e r (Davies, 1975) such crosses were used to e x a m i n e t h e role of cell n u m b e r , d r y a n d wet cell weight, g r o w t h rate, w a t e r c o n t e n t a n d g r o w t h period as d e t e r m i n a n t s of seed size a n d to e x a m i n e how t h e y are regulated. I n this r e p o r t studies of t h e nuclear D N A a n d of t h e r i b o s o m a l R N A (rRNA) contents of t h e c o t y l e d o n cells are described a n d t h e results considered in r e l a t i o n to t h e two s y s t e m s of control.
Materials and Methods Two varieties of Pisum sativum and their reciprocal hybrids were used; J I 430 (cv Greenshaft) and J I 181 (Keeran Pea from Nepal). The mean dry seed weights (in g) of J I 430,430 • 181,181 and 181 • 430 are 0.244:, 0.246, 0.073 and 0.111 respectively. DNA Contents. Tissues fixed in 3 parts ethanol: 1 part glacial acetic acid were hydrolysed in 5N HC1 at 20~ for 60 min, washed three times with distilled water and stained with Feulgen for 60 rain. They were then rinsed in fresh SO2 water and mounted on a coverslip in glycerol and covered with cellophane. This allowed the use of a crushing condenser to flatten the nuclei of the ee]ls prior to measuring their DNA content in a Vickers M85 microdensitometer. DNA values from at least 100 cells per sample were measured.
304
D. 1~. Davies and V. Brewster
Ribosomal RNA Contents. The amount of RNA in the cotyledons of J I 181 and J I 430 and their reciprocal hybrids was examined at different developmental stages and the proportions of different I~NAs determined in J I 181 and J I 430. To determine the proportions of 25s, 18s, 5s and 4s RNA at different stages, the I~NA was extracted by the method of Weinmann (1972) with the exception that 0,5% and 1.0% Sodium •-lauroyl sarcosinate was used as the detergent in their solutions I and I I I respectively. If double gels, 10% and 2,5% acrylamide, (Loening, 1967) were used for separating the I~NA species, it was impossible to accurately resolve the amount of 5s and 4s present without grossly overloading the gels for the other species of I~NA. Separate 2.5 % and 10 % gels were therefore used which could be loaded accordingly. Gels were run at 5 mA/gel at 4~ using Weinmann's (1972) buffer, after prerunning for 45 min. The gels were scanned in a Joyee-Locbl Chromoscan and the proportions of the different l ~ A s detected. Total RNA per cotyledon was routinely extracted either by the method of Click and Hackett (1966), and estimated by the orcinol method (Schneider, 1957) or by a modification of the method of Wilcoekson (1973) and estimated at OD 260. The estimates of RNA contents were identical with the two methods.
Results D N A Contents
Since t h e r e is a correlation b e t w e e n cell size a n d D N A c o n t e n t (Bennett, 1972) t h e nuclear D N A contents of t h e cotyledons of t h e varieties J I 181 a n d 430 were compared. These varieties have s u b s t a n t i a l different seed a n d cell weights (Davies, 1975). The results of t h e D N A e s t i m a t i o n s are shown in Fig. 1 t o g e t h e r w i t h d a t a from a r o o t t i p m e r i s t e m of J I 181. The l a t t e r has peaks representing the 2C a n d 4C values of G1 a n d G 2 cells r e s p e c t i v e l y A t 18 a n d 21 days, t h e cells of J I 181 h a d increased t h e i r D N A contents more r a p i d l y t h a n those of J I 430 b u t t h e r e was no clear difference b e t w e e n t h e two g e n o t y p e s thereafter. B o t h showed a wide s p r e a d of values u p to 64C, a n d a range of cell volumes was also observed. T h e a p p a r e n t r e d u c t i o n a t l a t e r stages (33 a n d 36 days) a l m o s t c e r t a i n l y is an a r t i f a c t arising from t h e difficulty of a d e q u a t e l y squashing t h e nuclei of t h e m o r e m a t u r e cells with t h e i r dense cellular contents. There is no evidence in these d a t a t h a t differences in t h e average cell weight of J I 181 a n d J I 430 can be a t t r i b u t e d to gross differences in D N A contents t h o u g h t h e resolution of t h e m e a s u r e m e n t s is such t h a t a slightly higher p r o p o r t i o n of larger cells a n d nuclei in J I 430 would n o t h a v e been detected. To d e t e c t this it would be necessary to s a m p l e s u b s t a n t i a l l y m o r e cells t h a n t h e ~ 100 scored in t h e p r e s e n t experiments. Ribosomal R N A Contents
The p r o p o r t i o n of 25s, 18s, 5s a n d 4s R N A d i d n o t differ b e t w e e n t h e g e n o t y p e s J I 181 a n d J I 430 or between t h e different stages e x a m i n e d from 18 d a y s to 27 d a y s after flowering. 25s plus 18s R N A c o n s t i t u t e d a p p r o x i m a t e l y 89 % of t h e t o t a l I~NA in J I 181 a n d 85% in J I 4 3 0 (Fig. 2). I n view of this, s u b s e q u e n t results are q u o t e d in t e r m s of t o t a l R N A a m o u n t s . The results for t h e R N A a m o u n t s are given in Fig. 3, p l o t t i n g t h e values a g a i n s t weights of c o t y l e d o n r a t h e r t h a n d a y s ; t h e p a t t e r n of results is t h e same with either b u t in c o m p a r i n g seed from different plants, t i m e is often a less a c c u r a t e d e t e r m i n a n t of d e v e l o p m e n t a l stage t h a n weight of cotyledon. A n e x p a n d e d scale is used for t h e J I 181 a n d J I 181 • 430 d a t a to p r e v e n t clustering of points, b u t t h e regression values shown, indicate t h a t
Studies of Seed Development in Pisum sativum
305
JI 181 JI 430
24 I
,
L
i
27 P
30
33qtoM;-, 4
6
8
np, ~
,
10
4
6
8
1]],
10
DNA/cell {log 2)
Fig. 1. DNA amounts (in arbitrary units) per cotyledon cell of JI 181 and JI 430, RT = root tips; 18 to 36 refers to developmental stage in number of days after flowering
the slopes of the lines are similar for all genotypes. The t~NA per unit weight of fresh cotyledons is constant for all genotypes and developmental stages examined (Fig. 4) even though this unit weight is made up different numbers of cells in the different genotypes. Since no cell division is occurring in the period depicted from 18 days after flowering onwards, the increase in RNA represents a very marked increase per cell. I n J I 4 3 0 and J I 4 3 0 • 181 this is approximately 9 fold (over 15 days), but in J I 181 and J I 181 • 430 only approximately 3 fold,--all having similar initial values at 18 days. I t is here that we see a clear cut distinction between small and large seeded types. There is first a marked difference in the final average amount of t~NA per cell, J I 430 having nearly 2.5 times as much R N A
306
D. R. Davies and V. Brewster
25s
A
18s
F i g . 2 A and B. Chromoscan traces of R:NA extracted from J I 430. (a) 2.5% acrylamide gel showing 25s a n d 18s peaks; (b) 10% acrylamide gel showing 5s and 4s peaks. Five times as much R~NA was located on to the 10% as onto the 2.5% gel
//~
10 . c80
Z~ 7:
o
ooOO
0
~
6-
o
X
e-
< 3 z a: 2
20 100
40 60 200 300 wet wt.in mg/cotytedon
80 (181f181xZ,30) 400 (430f130x181)
Fig. 3. RNA per cotyledon a~ different developmental stages. J I 430 o; J I 430 x 181 []; J I 181 O; J I 181 x 430 9 .8'
~7 ~6 o
h x 3
I0
L
