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Stored polyadenylated RNA and loss of vigour in germinating wheat embryos
Plant Science, 38 (1985) 71--79
71
Elsevier Scientific Publishers Ireland Ltd.
STORED P O L Y A D E N Y L A T E D R N A AND LOSS OF V I G O U R IN GERMINATING WHEAT EMBRYOS
CLIFFORD M. BRAY* and C.A. DALE SMITH**
Department of Biochemistry, University of Manchester, Oxford Road, Manchester, M13 9PT (U.K.) (Received July 3rd, 1984) (Accepted November 16th, 1984) Loss of vigour in wheat seed is associated with lesions affecting the rate of disappearance of stored polyA÷ RNA (presumptive mRNA) in the germinating embryo when germination takes place at a sub-optimal temperature. During germination in the presence of a-amanitin and consequent absence of de novo polyA+ RNA biosynthesis, the wheat embryo can degrade up to 70% of the stored polyA÷ RNA of the quiescent embryo before any significant reduction in the rate of protein biosynthesis in the embryo becomes apparent. It is possible that two subpopulations of polyA÷ RNA species exist in wheat embryos during early germination, one population being degraded rapidly upon rehydration of the embryo whilst the other population supports protein biosynthesis in the initial germination stages prior to degradation.
Key words: polyadenylated RNA; germination; wheat; vigour
Introduction A seed is considered viable when it possesses the capacity to germinate normally under the optimal conditions of the standard germination test [1,2]. Seed vigour, however, is a much more nebulous concept, differences in the vigour of seed lots of comparable viability being manifested in the capability for, and rapidity of, eventual seedling emergence under non-ideal conditions found in the field or the stress conditions employed to simulate field conditions in laboratory vigour tests. These differences due to loss of seed vigour in seed lots of high viability are n o t observed under optimal germination conditions b u t field trials have shown that seed vigour is a significant factor affecting establish-
*To whom correspondence should be sent. **Present address: Department of Health and Human Services, National Institutes of Health, National Cancer Institute, Bethesda, MD 20205, U.S.A. Abbreviations: mRNP, messenger ribonucleoprotein; polyA÷ RNA, presumptive mRNA.
ment, growth and final yield in cereals [3,4]. At a molecular level, loss of both viability and vigour is accompanied by a dramatic decline in protein and R N A biosynthetic activity [5--8] and can also be related to decreased mitochondrial activity [9] and defective ATP regenerative processes [10,11]. Recent studies have demonstrated that the vigour of a cereal seed lot is related to the rate of R N A and protein synthesis in the cereal e m b r y o during the early hours of germination [7]. Loss of vigour in cereals is also reflected in altered patterns o f polyA÷ R N A metabolism and polyA÷ R N A levels in the cereal e m b r y o particularly upon germination at a sub-optimal temperature [12,13]. Although the polyadenylated R N A species present in the quiescent e m b r y o prior to germination have been shown to be active in directing protein synthesis in cell-free systems [14,15] a definite in vivo role for this R N A in the germination process has been questioned [16] particularly as newly synthesised polyadenylated R N A species can be found associated with polysomes as early as 1.5 h after
0168-9452/85/$03.30 © Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
72 c o m m e n c e m e n t of imbibition in wheat embryos [17]. The work described here concerns a detailed study of the role of the pre-existing (long-lived) polyA÷ R N A species of the quiescent wheat e m b r y o in both the support of protein biosynthesis during the early germination period and upon the germinative vigour of the seed lot. This study has made use of a p o t e n t inhibitor of p o l y A÷ R N A synthesis, the fungal toxin a-amanitin, which has permitted a study of the temporal decay of pre-existing polyA÷ R N A species in the absence of de novo polyA÷ R N A synthesis in embryos from high and reduced vigour wheat seed during germination. Under these circumstances it is possible to estimate the fraction of stored polyA÷ R N A available for translation during germination and to determine whether loss of seed vigour is reflected in differences in the metabolism of stored polyA÷ R N A species in embryos from seed lots o f different vigour ratings. Materials and m e t h o d s
Plant materials and germination conditions Wheat (Triticum aestivum var. Hobbit) seed lots of differing viability and vigour ratings were supplied b y RHM Research Ltd., High Wycombe, Bucks., U.K. who performed the germination tests on the seed lots to determine their viability scores using the standard ISTA germination test. Vigour tests were also performed on the seed lots by RHM Research Ltd. using a stress germination test the details of which are not published for reasons of industrial confidentiality. The seed lots used had viability and vigour scores (on a 0--100 scale) of 89 viability, 91 vigour (high viability, high vigour); 85 viability, 69 vigour (high viability, reduced vigour). For the purposes of clarity these seed lots will be referred to as high vigour and medium vigour seed lots, respectively. Wheat embryos were prepared from these seed lots b y the method of Johnston and Stern [18] and germinated as previously described [7].
RNA extraction and quantitation of polyadenylated R N A (polyA ÷RNA ) After germination for the required time, embryos (50) were blotted dry and either used immediately or frozen and stored under liquid nitrogen until required. Total cellular RNA was extracted by the pH 9-SLS-phenol method [19] and polyA÷ R N A content estimated by oligo dT-cellulose chromatography [30] or hybridisation with [3 H] polyuridylic acid [21] as previously described [ 13 ]. Imbibition conditions for pulse-labelling experiments After germination of embryos for the required time pulse-labelling of either R N A using [ 5 -3 H ] uridine or protein using [ U -14C ] amino acid mixture in the germination medium was performed as described previously [ 13 ]. Results
R N A synthesis in wheat embryos during loss of vigour The proportion of RNA synthesised in the wheat e m b r y o which becomes polyadenylated varies significantly during the early hours of germination (Table I). In addition, the rate of RNA synthesis in embryos germinated at 20 ° C is much higher (2--4-fold) than in embryos germinated at 10°C. Whereas no difference can be seen between the rate of incorporation of [3 H] uridine into total cellular RNA in high or medium vigour embryos at 20°C except possibly during the initial 2.5 h of germination, a significant reduction in the rate of incorporation is seen in medium vigour embryos compared with high vigour embryos when germination takes place at 10°C. Total R N A synthesis in medium vigour embryos during the first hour of germination at 10°C is 42% of the rate in high vigour embryos whilst at 17--18 h of germination this rate has risen to 72% of the level of the high vigour controls. In both high vigour embryos germinated at either an optimal germination temperature (20°C) or stress temperature (10°C) and medium vigour embryos germinated at 20°C
Total RNA p o l y A÷ R N A
Total RNA p o l y A÷ R N A
Total RNA p o l y A÷ R N A
1.5--2.5
6.5--7.5
17--18
Total RNA p o l y A+ R N A
Total RNA p o l y A÷ R N A
Total RNA p o l y A+ R N A
Total RNA p o l y A+ R N A
0--1
1.5--2.5
6.7--7.5
17--18
Medium vigour
Total RNA p o l y A÷ R N A
RNA fraction
0--1
High vigour
Pulselabelling p e r i o d (h)
48711 8593 (17.6) 53228 7459 (14.0) 110112 12443 (11.2) 193160 13714 (7.1)
54744 11077 (20.2) 71359 11801 (16.5) 106169 13435 (12.7) 184530 12456 (6.7)
2 6 3 2 9 -+ 2 0 1 4 1 5 3 6 _+3 5 9 2 3 6 7 4 _+ 1041 827 -+ 120 4 5 2 4 6 +__4 4 1 3 6 3 2 +_ 99 7 5 3 5 3 _+ 5 4 1 2 5 5 9 _+ 101
_+3010 -+ 6 9 8 _+ 2 3 2 9 -I-2413 _+ 7 4 5 2 -+ 1 6 0 5
7 5 6 0 6 -+ 9 3 1 3 598 -+ 102
4 7 7 0 6 _+ 3 1 4 3 1 2 1 4 _+3 0 4
3 3 8 3 9 _+ 2 2 2 3 1 7 7 0 -+ 106
2 2 0 9 8 -+ 1113 1487 Jr 4 4 1
c.p.m, r e c o v e r e d with a-amanitin (mg-' RNA) (20°C)
"1-8 9 9 +_ 2 9 9
-+ 1 0 1 5 3 +_ 1 1 3 8
-+ 4 7 5 9 _+ 1 7 1 2
_+ 2 2 1 2 -+ 1 0 3 7
-+ 6 2 4 1 _+ 1 5 4 4
c.p.m, r e c o v e r e d in c o n t r o l (mg-' RNA) (20°C)
61 96
59 93
50 89
54 82
59 95
55 91
53 85
60 83
% inhibition (20°C)
8025 788 (9.8) 18871 1703 (9.0) 31143 3209 (10.3) 45593 4154 (9.1)
18955 3581 (18.9) 32340 3851 (11.9) 45490 5141 (11.3) 62768 4713 (7.5)
"1- 1 3 5 9 _+ 777
-+ 3 7 8 9 -+ 1 7 2
+_4116 _+ 3 5 4
"1"7 5 2 _+ 110
-+ 3 7 9 9 -+ 8 1 9
_+ 2 1 1 8 _+ 717
+_ 2 7 7 6 -+ 9 1 0
_+ 1 2 5 4 -+ 8 0 9
c.p.m, r e c o v e r e d in c o n t r o l (rag-' RNA) (10°C)
7 3 1 5 -+ 1071 235 "1-62
9 6 6 1 -I" 1 1 2 8 253 -+ 63
9 0 9 3 -+ 1 0 5 9 98 _+31
4 2 3 4 + 711 134 + 75
2 0 7 3 3 _+7 1 2 4 9 3 +_ 70
2 2 7 2 3 _+ 2 8 6 4 187 _+ 38
1 6 4 7 3 -+ 1321 2 9 2 -+ 51
7 6 0 3 _+ 210 493 _+ 99
c.p.m, r e c o v e r e d with a-amanitin (mg ' R N A ) (10°C)
84 94
69 92
52 94
47 83
67 90
50 90
49 92
60 80
% inhibition (10°C)
T a b l e I. E f f e c t o f ~ - a m a n i t i n o n R N A s y n t h e s i s in g e r m i n a t i n g w h e a t e m b r y o s d u r i n g loss o f vigour. E m b r y o s were pulse-labelled using [5 -3 H ] uridine for 1 h at t h e t i m e s i n d i c a t e d at 20°C o r 10°C in t h e p r e s e n c e or a b s e n c e o f 12 u M ~ - a m a n i t i n . T o t a l cellular R N A was e x t r a c t e d as described a n d f r a c t i o n a t e d b y oligo dT-cellulose c h r o m a t o g r a p h y . Results r e p r e s e n t c o u n t s r e c o v e r a b l e m g ' R N A in all R N A fractions ( t o t a l R N A ) or in f r a c t i o n III ( p o l y A + R N A ) a f t e r oligo dT-cellulose c h r o m a t o g r a p h y . Results r e p r e s e n t the average of d u p l i c a t e d e t e r m i n a t i o n s . Figures in p a r e n t h e s e s r e p r e s e n t t h e r a d i o a c t i v i t y r e c o v e r e d in t h e p o l y A ÷ R N A f r a c t i o n expressed as a p e r c e n t a g e o f c.p.m, recovered in t o t a l cellular RNA.
--3
74 the proportion of RNA synthesised which is polyadenylated falls steadily from 18--20% of the total RNA synthesised during the first hour of germination to approx. 7% at 17--18 h of germination (Table I). This trend is not observed in medium vigour embryos germinated at 10°C. At this sub-optimal germination temperature the proportion of polyA÷ RNA synthesised in medium vigour embryos does n o t vary and accounts for 9--10% of all RNA species synthesised at all germination times studied during a 0--18-h germination period. a-Amanitin, an inhibitor of eukaryotic RNA polymerase II is a very potent inhibitor of polyA÷ RNA synthesis in high and medium vigour embryos during germination at either 20°C or 10°C but is much less effective in the inhibition of total cellular RNA synthesis (Table I). The inhibitory effect of a-amanitin on polyA÷ RNA synthesis in germinating wheat embryos is maximal at a concentration of 12 gM a-amanitin in the germination medium (results not shown) and this inhibitory concentration was used in experiments to study the decay of stored polyA÷ RNA in germinating wheat embryos in the absence of de novo polyA÷ RNA synthesis. Stored m R N A levels in wheat embryos In high vigour embryos germinated at 20°C in the absence of a-amanitin the level of polyA÷ RNA falls dramatically during the first hour of germination, remains constant during the 1--3 h germination period, then increases rapidly up to a level similar to that found in quiescent embryos by 7 h of germination (Fig. 1A). Medium vigour embryos germinated at 20°C show similar trends in polyA÷ RNA metabolism although the initial decrease and subsequent increase in polyA÷ RNA levels are less marked (Fig. 1A). Upon germination of wheat embryos at a stress temperature (10°C) significant changes in polyA÷ RNA metabolism occur in both high and medium vigour embryos compared with the changes observed at 20°C, the differences being minor in the case of high vigour embryos but major for medium vigour embryos (Fig. 1B). At 10°C, high vigour
embryo polyA ÷ RNA levels decrease much less during the first hour of germination than at 20°C but exhibit the same general trend of a decline in polyA÷ RNA content followed by an increase back to the original level found in quiescent embryos over 0--7 h of germination. In medium vigour embryos germinated at 10°C the level of polyA÷ RNA i n t h e embryo remains unchanged for up to 3 h of germination before declining to a level of about half that found in high vigour quiescent embryos. This level then changes very little up to 24 h of germination (Fig. 1B). As the total a m o u n t of extractable RNA is similar in high and reduced vigour embryos over the time period studied the relative changes in levels of polyA÷ RNA are a true indication of the differences in polyA÷ RNA contents as a proportion of total cellular RNA and can be assumed to reflect levels of presumptive messenger RNA present in these embryos at the different imbibition stages. Inhibition of polyA÷ RNA synthesis in wheat embryos during germination in the presence of 12 pM a-amanitin permits a study of the disappearance of stored polyA÷ RNA (stored potential mRNA) species during germination. The results of these studies are presented in Fig. 2. During germination of high and medium vigour embryos at 20°C in the presence of a-amanitin the disappearance of stored polyA÷ RNA in these embryos follows similar patterns {Fig. 2A,C). An initial sharp decline in polyA÷ RNA levels over the 0- to 1-h germination period to reach a level of polyA÷ RNA which is approximately 40% of that found in the quiescent embryo reflects the steep decline in polyA÷ RNA content observed during germination at 20°C in the absence of a-amanitin (Fig. 1A). However, upon continuation of germination in the presence of ~amanitin beyond I h, no subsequent increase in polyA÷ RNA levels is observed (Fig. 2A,C} and the level of polyA÷ RNA in high and medium vigour embryos continues to decline up to 8 h of germination at a significantly reduced rate compared with the decline in the first hour of germination. After this time little
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Fig. 1. Embryos were germinated for the required time at 20°C (A) or 10°C (B) and total cellular RN A extracted as described. PolyA÷ R N A content was estimated as described in the Materials and methods section. Results represent the average +_ S.E.M. of 6 independent determinations. The 100% level of c.p.m, bound, i.e. the quiescent high vigour embryo value, is equivalent to 96 ng poly A ÷ R N A / 1 0 ug total RNA sample. The remaining time points are expressed as a percentage of c.p.m, bound by R N A from quiescent embryos high vigour (I I); medium vigour ( I - - - - - - I ) .
p o l y A÷ R N A remains in the ~-amanitin-treated embryos. Slight differences in the rate of decay o f polyA÷ R N A in high and medium vigour embryos at 20°C are reflected in the half-life of the decay of polyA÷ RNA, this being 0.35 h in high vigour embryos but 1.2 h in medium vigour embryos. Upon germination at 10°C in the presence of 12 ~M ~-amanitin distinct differences are seen in the decay profiles of stored polyA÷ R N A in high and medium vigour embryos (Fig. 2B,D). The degradation of polyA÷ R N A in high vigour embryos commences without any apparent delay although the decay of polyA÷ R N A at 10°C is slightly slower, with a half-life of 1.6 h compared with 0.35 h at 20°C. Again, there is little polyA÷ R N A de-
tectable in high vigour embryos after 6--8 h of germination. However, in medium vigour embryos there is a pronounced delay before any significant reduction in the level of p o l y A÷ R N A in these embryos is observed (Fig. 2B,D) and only after 2 h of germination does the level of p o l y A÷ R N A in medium vigour embryos decline significantly reaching levels that are barely detectable by 8 h of germination. In contrast during the initial 2 h germination period the level of p o l y A ÷ RNA in high vigour embryos germinated in the presence of ~-amanitin falls by 50%. This initial delay period in polyA ÷ RNA decay in the presence of ~-amanitin is also seen in medium vigour embryos germinated at 10°C in the absence of ~-amanitin (Fig. 1B).
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HOURS OF IMBIBtTION Fig. 2. S t o r e d m R N A - d i r e c t e d p r o t e i n s y n t h e s i s in high a n d m e d i u m vigour w h e a t e m b r y o s . E m b r y o s were pulse labelled for 1 h at t h e t i m e s i n d i c a t e d using [ 1 4 C ] a m i n o acid m i x t u r e in t h e p r e s e n c e or a b s e n c e o f 12 # M aa m a n i t i n . T h e u p t a k e a n d i n c o r p o r a t i o n o f [ 1 4 C ] a m i n o acids was d e t e r m i n e d as d e s c r i b e d in t h e text. The results are p r e s e n t e d as a h i s t o g r a m a n d r e p r e s e n t t h e average + S.E.M. o f 3 i n d e p e n d e n t e x p e r i m e n t s . The ' 1 0 0 % level' o f [ '4C 1 a m i n o acid i n c o r p o r a t i o n is t a k e n as t h e level o f i n c o r p o r a t i o n o f [ '4C] a m i n o acids i n t o acid-precipitable m a t e r i a l in t h e 0--1-h pulse p e r i o d a f t e r c o r r e c t i o n for a n y d i f f e r e n c e s in u p t a k e o f r a d i o a c t i v e a m i n o acids by the e m b r y o s u n d e r d i f f e r e n t g e r m i n a t i o n c o n d i t i o n s [ 7 ] a n d was e q u a l to 1025 +_ 10 c.p.m, i n c o r p o r a t e d i n t o acidp r e c i p i t a b l e m a t e r i a l p e r e m b r y o . This value was t h e s a m e w h e t h e r a - a m a n i t i n was p r e s e n t or a b s e n t from t h e germ i n a t i o n m e d i u m d u r i n g t h e initial 0--1-h pulse period. T h e curves (-- - - --) r e p r e s e n t the d e c a y o f s t o r e d p o l y A + R N A species in w h e a t e m b r y o s d u r i n g g e r m i n a t i o n in t h e p r e s e n c e o f 12 # M ~ - a m a n i t i n at 20°C o r 10°C. P o l y A ÷ R N A levels were d e t e r m i n e d a n d e x p r e s s e d as d e s c r i b e d in t h e legend to Fig. 1.
Stored messenger I~NA and protein biosynthesis When e m b r y o s are germinated at 20°C in the absence of a-amanitin their in vivo rate of protein biosynthesis increases over the initial 7-h germination period [7]. During this period e m b r y o s of high vigour synthesise protein at higher rates than medium vigour embryos. However, b y 17 to 18 h of germination at 20°C high and medium vigour embryos synthesise protein at similar rates [7]. Germination of embryos at a stress germination temperature (10°C) results in a much lower rate of protein biosynthesis in both high and medium vigour embryos than is observed at 20°C. In addition high vigour embryos synthesise protein at higher rates than medium
vigour embryos throughout the initial 18-h germination period at 10°C [7]. Although the rapid increase in protein synthetic rate during germination is not seen in embryos germinated in ~-amanitin (Fig. 2), the rate of protein synthesis in these embryos does not reflect the falling level of polyA÷ RNA {potential m R N A ) species within the embryos. Upon germination of high vigour embryos at 20°C in the presence of~-amanitin the level of polyA÷ R N A in the embryo falls rapidly yet it is not until approx. 80% of the polyA÷ R N A found in quiescent high vigour embryos has been degraded that any significant decrease in protein biosynthetic rate in the embryos is observed {Fig. 2A). A similar situation is seen in embryos of medium vigour ger-
77 minated at 20°C. Although the initial rates of protein synthesis are lower in these embryos than in high vigour embryos, no reduction in in vivo protein synthetic rates is seen in medium vigour embryos, even though polyA÷ R N A levels fall dramatically over the first 3 h of germination, until approx. 70--80% of the polyA÷ R N A found in quiescent embryos of medium vigour has been degraded (Fig. 2C). After this time (2--3 h of germination) the rate of protein synthesis in medium vigour embryos decreases sharply. Upon germination of embryos at 10°C in the presence of ~-amanitin, high vigour embryos exhibit a short lag period {0--1 h) when protein synthesis occurs at a very low rate {Fig. 2B} before synthesising protein at 60--70% of the rate found in high vigour embryos at 20°C between 1--3 h of germination. During this period polyA+ R N A levels in the e m b r y o fall to 30% of the level found in quiescent high vigour embryos. It is only when the level of polyA÷ R N A falls below this level, i.e. from 3 h of germination onwards, than any significant decrease in the rate of protein biosynthesis is observed {Fig. 2B). When medium vigour embryos are germinated in the presence of ~-amanitin at 10°C an initial lag period (0 -1 h) during which protein synthesis is barely detectable is followed by a 3-h period where little difference is noted in the protein synthetic capacity of the embryos even though the level of polyA+ R N A in these embryos declines to about 40% of the level in quisecent medium vigour embryos (Fig. 2D). After this time, however, the continuing slow decay of polyA÷ R N A levels in the e m b r y o s is accompanied by a significant decrease in the in vivo protein synthetic rate. Throughout the initial 4 h of germination at 10°C in the presence of a-amanitin the rate of protein synthesis in high vigour embryos is significantly greater than the level in medium vigour embryos. Discussion Quiescent wheat embryos contain polyA÷ RNA species which are degraded rapidly during
the initial stages of germination to be replaced b y polyA÷ R N A species which are produced during germination as a result of de novo R N A biosynthesis. The results presented here suggest that germinating wheat embryos can degrade up to 60--70% of this stored polyA÷ R N A before any significant reduction in the rate of in vivo protein biosynthesis directed b y this polyA÷ R N A becomes apparent {Fig. 2 ). This observation suggests that only 30--40% of this stored polyA÷ R N A is available to the protein synthesising apparatus of the embryo for translation into protein during early germination. It is possible that t w o different sub-populations of polyA÷ R N A species exist in the quiescent e m b r y o , one population being actively involved in the initiation of protein synthesis during seed germination whilst the other population may be merely 'carried over' from the ripening period and would comprise that proportion of the polyA÷ R N A population which disappears rapidly upon germination without affecting the in vivo rate of protein biosynthesis in the e m b r y o (Fig. 2). If this is the case, the germinating wheat embryo must possess a mechanism whereby it can distinguish those polyA÷ RNA species which are to be degraded during the first 2--3 h of germination from those species which are to be conserved to support protein biosynthesis during this time. The answer to h o w this distinction can be achieved may lie not in the polyA÷ R N A per se b u t in the nature of the proteins which are associated with the polyA÷ R N A since in the cell the polyA÷ R N A species complex with specific proteins to form messenger ribonucleoprotein {mRNP) particles. Control of the onset of protein biosynthesis during the early hours of germination of wheat embryos may be analogous to the situation which exists during early embryonic development following fertilisation of echinoderm and amphibian eggs [22]. In these developing e m b r y o s it has been suggested that the translation of maternal m R N A species, which was repressed in the unfertilised egg, is switched on after fertilisation by a mechanism as yet u n k n o w n b u t which
78 m a y involve a role for the proteins specifically associated with the mRNP particles [23]. Different classes o f m R N P particles can be isolated f r o m several o t h e r eukaryotic sources, b o t h animal [24] and plants [25]. In the germinating wheat e m b r y o different size classes o f native m R N P particles have been shown to exist [25] and the half-life of the template RNA species associated with these particles is o f the order o f 2 h at optimal germination t e m p er atu r es [26]. Thus, it is possible t hat some m R N P species in the quiescent wheat e m b r y o are pre-destined for rapid degradation during early germination whilst o t h e r species (30--40% o f the population) are actively involved in the initiation of protein synthesis during germination, their fate being controlled, at least partially, by the nature o f the specific protein c o m p o n e n t s associated with t he different m R N P particles. Loss o f vigour in wheat seed can be correlated with p o ly A÷ RNA levels in the e m b r y o and the rate of protein biosynthesis in these e m b r y o s after 24 h of germination at 10°C [12,13]. Indeed the rate of in vivo protein biosynthesis in the e m b r y o at this time is indicative o f the vigour rating of the seed lot, whereas this correlation is not seen between rates of total RNA synthesis and the vigour o f t h e e m b r y o s [13]. A detailed study of the stored m R N A c o n t e n t of e m br yos f r om two o f t h e seed lots used in previous studies on loss o f vigour [13] is r e p o r t e d here and indicates t h a t a fraction of this stored m R N A is capable o f supporting protein synthesis in the e m b r y o during the period immediately following reh y d r a t i o n o f the embryos, even in the absence of de novo p o ly A÷ RNA synthesis, Loss of vigour o f a seed lot in the absence of any significant loss of viability is associated with a change in the p atte r n of decay o f stored messenger RNA in reduced vigour e m br yos germinated under sub-optimal conditions (Fig. 2B,D). However, little difference is seen in p o l y A÷ RNA decay patterns in high and m e d i u m vigour e m b r y o s germinated at 20°C, a result which correlates with observations demonstrating th at vigour differences between
seed lots are not observable under optimal germination conditions [2,4]. The molecular basis for these differences in polyA÷ RNA metabolism in seed lots of varying vigour during germination at sub-optimal temperatures is not know n but current investigations in this l a b o r a t o r y are aimed at understanding the factors controlling translatability, conservation and decay of potential m R N A species during germination and the importance of the lesions appearing in m R N A turnover during loss of vigour, and subsequently loss of viability in cereals. Acknowledgements The financial support of the Lord Rank Prize Funds is gratefully acknowledged. References 1 E.H. Roberts, Viability of seeds, E.H. Roberts (Ed.), Chapman and Hall Ltd. 1972. 2 D.A. Stormonth, Developments in the business and practice of cereal seed trading and technology. pp. 49--76, P.R. Hayward (Ed.), The Gavin Press, London, 1978. 3 D.A. Stormonth and D. Doling, Arable Farming, 6 (1979) 42. 4 J.G. Hampton, N.Z.J. Exp. Agric., 9 (1981) 191. 5 S. Sen and D.J. Osborne, Biochem. J. 166 (1977) 33. 6 C.M. Bray, Recent advances in the biochemistry of cereals, D.L. Laidman and R.G. WymJones (Eds.), Academic Press, New York, 1979, pp 147--173. 7 L.E. Blowers, D.A. Stormonth and C.M. Bray, Planta, 150 (1980) 19. 8 W.J. Peumans and A.R. Carlier, Biochem. Biophys. Pflanz., 176 (1981) 384. 9 S.S. Abu-Shakra and T.M. Ching, Crop Sci., 7 (1967) 115. 10 T.M. Ching, Plant Physiol., 51 (1973) 400. 11 S.A. Standard, D. Perret and C.M. Bray, J. Exp. Bot., 34 (1983) 1047. 12 C.A.D. Smith and C.M. Bray, Planta, 176 (1982) 413. 13 C.A.D. Smith and C.M. Bray, Plant Sci. Letts., 34 (1984) 335. 14 S. Spiegel and A. Marcus, Nature, 256 (1975)228. 15 M.E. Gordon and P.I. Payne, Planta, 130 (1976) 269. 16 P.I. Payne, Biol. Rev., 51 {1976) 329.
79 17 S. Spiegel, R.L. Obendorf and A. Marcus, Plant Physiol., 56 (1975) 502. 18 F.B. Johnston and H. Stern, Nature, 74 {1957) 170. 19 G. Brawerman, Methods Enzymol., 30 (1974) 605. 20 M. Aviv and P. Leder, Proc. Natl. Acad. Sci. (Wash.) 69 (1972) 1408. 21 M. Rosbash and D.J. Ford, J. Mol. Biol., 85 (1974) 87. 22 A. Marcus, S. Spiegel and J.D. Brooker, Control Mechanisms in Development, R.H. Meints and E.
23 24 25 26
Davies (Eds.), Plenum Press, New York and London, 1975, pp. 1--20. J.D. Richter and L.D. Smith, Nature, 309 (1984) 378. H.P. Schmidt, K. Kohler and B. Setyono, Mol. Biol. Rep., 9 (1983) 87. W.J. Peumans and A.R. Carlier, Planta, 136 (1977) 195. L.I. Caers, W.J. Peumans and A.R. Carlie~', Pianta, 144 (1979) 491.
71
Elsevier Scientific Publishers Ireland Ltd.
STORED P O L Y A D E N Y L A T E D R N A AND LOSS OF V I G O U R IN GERMINATING WHEAT EMBRYOS
CLIFFORD M. BRAY* and C.A. DALE SMITH**
Department of Biochemistry, University of Manchester, Oxford Road, Manchester, M13 9PT (U.K.) (Received July 3rd, 1984) (Accepted November 16th, 1984) Loss of vigour in wheat seed is associated with lesions affecting the rate of disappearance of stored polyA÷ RNA (presumptive mRNA) in the germinating embryo when germination takes place at a sub-optimal temperature. During germination in the presence of a-amanitin and consequent absence of de novo polyA+ RNA biosynthesis, the wheat embryo can degrade up to 70% of the stored polyA÷ RNA of the quiescent embryo before any significant reduction in the rate of protein biosynthesis in the embryo becomes apparent. It is possible that two subpopulations of polyA÷ RNA species exist in wheat embryos during early germination, one population being degraded rapidly upon rehydration of the embryo whilst the other population supports protein biosynthesis in the initial germination stages prior to degradation.
Key words: polyadenylated RNA; germination; wheat; vigour
Introduction A seed is considered viable when it possesses the capacity to germinate normally under the optimal conditions of the standard germination test [1,2]. Seed vigour, however, is a much more nebulous concept, differences in the vigour of seed lots of comparable viability being manifested in the capability for, and rapidity of, eventual seedling emergence under non-ideal conditions found in the field or the stress conditions employed to simulate field conditions in laboratory vigour tests. These differences due to loss of seed vigour in seed lots of high viability are n o t observed under optimal germination conditions b u t field trials have shown that seed vigour is a significant factor affecting establish-
*To whom correspondence should be sent. **Present address: Department of Health and Human Services, National Institutes of Health, National Cancer Institute, Bethesda, MD 20205, U.S.A. Abbreviations: mRNP, messenger ribonucleoprotein; polyA÷ RNA, presumptive mRNA.
ment, growth and final yield in cereals [3,4]. At a molecular level, loss of both viability and vigour is accompanied by a dramatic decline in protein and R N A biosynthetic activity [5--8] and can also be related to decreased mitochondrial activity [9] and defective ATP regenerative processes [10,11]. Recent studies have demonstrated that the vigour of a cereal seed lot is related to the rate of R N A and protein synthesis in the cereal e m b r y o during the early hours of germination [7]. Loss of vigour in cereals is also reflected in altered patterns o f polyA÷ R N A metabolism and polyA÷ R N A levels in the cereal e m b r y o particularly upon germination at a sub-optimal temperature [12,13]. Although the polyadenylated R N A species present in the quiescent e m b r y o prior to germination have been shown to be active in directing protein synthesis in cell-free systems [14,15] a definite in vivo role for this R N A in the germination process has been questioned [16] particularly as newly synthesised polyadenylated R N A species can be found associated with polysomes as early as 1.5 h after
0168-9452/85/$03.30 © Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
72 c o m m e n c e m e n t of imbibition in wheat embryos [17]. The work described here concerns a detailed study of the role of the pre-existing (long-lived) polyA÷ R N A species of the quiescent wheat e m b r y o in both the support of protein biosynthesis during the early germination period and upon the germinative vigour of the seed lot. This study has made use of a p o t e n t inhibitor of p o l y A÷ R N A synthesis, the fungal toxin a-amanitin, which has permitted a study of the temporal decay of pre-existing polyA÷ R N A species in the absence of de novo polyA÷ R N A synthesis in embryos from high and reduced vigour wheat seed during germination. Under these circumstances it is possible to estimate the fraction of stored polyA÷ R N A available for translation during germination and to determine whether loss of seed vigour is reflected in differences in the metabolism of stored polyA÷ R N A species in embryos from seed lots o f different vigour ratings. Materials and m e t h o d s
Plant materials and germination conditions Wheat (Triticum aestivum var. Hobbit) seed lots of differing viability and vigour ratings were supplied b y RHM Research Ltd., High Wycombe, Bucks., U.K. who performed the germination tests on the seed lots to determine their viability scores using the standard ISTA germination test. Vigour tests were also performed on the seed lots by RHM Research Ltd. using a stress germination test the details of which are not published for reasons of industrial confidentiality. The seed lots used had viability and vigour scores (on a 0--100 scale) of 89 viability, 91 vigour (high viability, high vigour); 85 viability, 69 vigour (high viability, reduced vigour). For the purposes of clarity these seed lots will be referred to as high vigour and medium vigour seed lots, respectively. Wheat embryos were prepared from these seed lots b y the method of Johnston and Stern [18] and germinated as previously described [7].
RNA extraction and quantitation of polyadenylated R N A (polyA ÷RNA ) After germination for the required time, embryos (50) were blotted dry and either used immediately or frozen and stored under liquid nitrogen until required. Total cellular RNA was extracted by the pH 9-SLS-phenol method [19] and polyA÷ R N A content estimated by oligo dT-cellulose chromatography [30] or hybridisation with [3 H] polyuridylic acid [21] as previously described [ 13 ]. Imbibition conditions for pulse-labelling experiments After germination of embryos for the required time pulse-labelling of either R N A using [ 5 -3 H ] uridine or protein using [ U -14C ] amino acid mixture in the germination medium was performed as described previously [ 13 ]. Results
R N A synthesis in wheat embryos during loss of vigour The proportion of RNA synthesised in the wheat e m b r y o which becomes polyadenylated varies significantly during the early hours of germination (Table I). In addition, the rate of RNA synthesis in embryos germinated at 20 ° C is much higher (2--4-fold) than in embryos germinated at 10°C. Whereas no difference can be seen between the rate of incorporation of [3 H] uridine into total cellular RNA in high or medium vigour embryos at 20°C except possibly during the initial 2.5 h of germination, a significant reduction in the rate of incorporation is seen in medium vigour embryos compared with high vigour embryos when germination takes place at 10°C. Total R N A synthesis in medium vigour embryos during the first hour of germination at 10°C is 42% of the rate in high vigour embryos whilst at 17--18 h of germination this rate has risen to 72% of the level of the high vigour controls. In both high vigour embryos germinated at either an optimal germination temperature (20°C) or stress temperature (10°C) and medium vigour embryos germinated at 20°C
Total RNA p o l y A÷ R N A
Total RNA p o l y A÷ R N A
Total RNA p o l y A÷ R N A
1.5--2.5
6.5--7.5
17--18
Total RNA p o l y A+ R N A
Total RNA p o l y A÷ R N A
Total RNA p o l y A+ R N A
Total RNA p o l y A+ R N A
0--1
1.5--2.5
6.7--7.5
17--18
Medium vigour
Total RNA p o l y A÷ R N A
RNA fraction
0--1
High vigour
Pulselabelling p e r i o d (h)
48711 8593 (17.6) 53228 7459 (14.0) 110112 12443 (11.2) 193160 13714 (7.1)
54744 11077 (20.2) 71359 11801 (16.5) 106169 13435 (12.7) 184530 12456 (6.7)
2 6 3 2 9 -+ 2 0 1 4 1 5 3 6 _+3 5 9 2 3 6 7 4 _+ 1041 827 -+ 120 4 5 2 4 6 +__4 4 1 3 6 3 2 +_ 99 7 5 3 5 3 _+ 5 4 1 2 5 5 9 _+ 101
_+3010 -+ 6 9 8 _+ 2 3 2 9 -I-2413 _+ 7 4 5 2 -+ 1 6 0 5
7 5 6 0 6 -+ 9 3 1 3 598 -+ 102
4 7 7 0 6 _+ 3 1 4 3 1 2 1 4 _+3 0 4
3 3 8 3 9 _+ 2 2 2 3 1 7 7 0 -+ 106
2 2 0 9 8 -+ 1113 1487 Jr 4 4 1
c.p.m, r e c o v e r e d with a-amanitin (mg-' RNA) (20°C)
"1-8 9 9 +_ 2 9 9
-+ 1 0 1 5 3 +_ 1 1 3 8
-+ 4 7 5 9 _+ 1 7 1 2
_+ 2 2 1 2 -+ 1 0 3 7
-+ 6 2 4 1 _+ 1 5 4 4
c.p.m, r e c o v e r e d in c o n t r o l (mg-' RNA) (20°C)
61 96
59 93
50 89
54 82
59 95
55 91
53 85
60 83
% inhibition (20°C)
8025 788 (9.8) 18871 1703 (9.0) 31143 3209 (10.3) 45593 4154 (9.1)
18955 3581 (18.9) 32340 3851 (11.9) 45490 5141 (11.3) 62768 4713 (7.5)
"1- 1 3 5 9 _+ 777
-+ 3 7 8 9 -+ 1 7 2
+_4116 _+ 3 5 4
"1"7 5 2 _+ 110
-+ 3 7 9 9 -+ 8 1 9
_+ 2 1 1 8 _+ 717
+_ 2 7 7 6 -+ 9 1 0
_+ 1 2 5 4 -+ 8 0 9
c.p.m, r e c o v e r e d in c o n t r o l (rag-' RNA) (10°C)
7 3 1 5 -+ 1071 235 "1-62
9 6 6 1 -I" 1 1 2 8 253 -+ 63
9 0 9 3 -+ 1 0 5 9 98 _+31
4 2 3 4 + 711 134 + 75
2 0 7 3 3 _+7 1 2 4 9 3 +_ 70
2 2 7 2 3 _+ 2 8 6 4 187 _+ 38
1 6 4 7 3 -+ 1321 2 9 2 -+ 51
7 6 0 3 _+ 210 493 _+ 99
c.p.m, r e c o v e r e d with a-amanitin (mg ' R N A ) (10°C)
84 94
69 92
52 94
47 83
67 90
50 90
49 92
60 80
% inhibition (10°C)
T a b l e I. E f f e c t o f ~ - a m a n i t i n o n R N A s y n t h e s i s in g e r m i n a t i n g w h e a t e m b r y o s d u r i n g loss o f vigour. E m b r y o s were pulse-labelled using [5 -3 H ] uridine for 1 h at t h e t i m e s i n d i c a t e d at 20°C o r 10°C in t h e p r e s e n c e or a b s e n c e o f 12 u M ~ - a m a n i t i n . T o t a l cellular R N A was e x t r a c t e d as described a n d f r a c t i o n a t e d b y oligo dT-cellulose c h r o m a t o g r a p h y . Results r e p r e s e n t c o u n t s r e c o v e r a b l e m g ' R N A in all R N A fractions ( t o t a l R N A ) or in f r a c t i o n III ( p o l y A + R N A ) a f t e r oligo dT-cellulose c h r o m a t o g r a p h y . Results r e p r e s e n t the average of d u p l i c a t e d e t e r m i n a t i o n s . Figures in p a r e n t h e s e s r e p r e s e n t t h e r a d i o a c t i v i t y r e c o v e r e d in t h e p o l y A ÷ R N A f r a c t i o n expressed as a p e r c e n t a g e o f c.p.m, recovered in t o t a l cellular RNA.
--3
74 the proportion of RNA synthesised which is polyadenylated falls steadily from 18--20% of the total RNA synthesised during the first hour of germination to approx. 7% at 17--18 h of germination (Table I). This trend is not observed in medium vigour embryos germinated at 10°C. At this sub-optimal germination temperature the proportion of polyA÷ RNA synthesised in medium vigour embryos does n o t vary and accounts for 9--10% of all RNA species synthesised at all germination times studied during a 0--18-h germination period. a-Amanitin, an inhibitor of eukaryotic RNA polymerase II is a very potent inhibitor of polyA÷ RNA synthesis in high and medium vigour embryos during germination at either 20°C or 10°C but is much less effective in the inhibition of total cellular RNA synthesis (Table I). The inhibitory effect of a-amanitin on polyA÷ RNA synthesis in germinating wheat embryos is maximal at a concentration of 12 gM a-amanitin in the germination medium (results not shown) and this inhibitory concentration was used in experiments to study the decay of stored polyA÷ RNA in germinating wheat embryos in the absence of de novo polyA÷ RNA synthesis. Stored m R N A levels in wheat embryos In high vigour embryos germinated at 20°C in the absence of a-amanitin the level of polyA÷ RNA falls dramatically during the first hour of germination, remains constant during the 1--3 h germination period, then increases rapidly up to a level similar to that found in quiescent embryos by 7 h of germination (Fig. 1A). Medium vigour embryos germinated at 20°C show similar trends in polyA÷ RNA metabolism although the initial decrease and subsequent increase in polyA÷ RNA levels are less marked (Fig. 1A). Upon germination of wheat embryos at a stress temperature (10°C) significant changes in polyA÷ RNA metabolism occur in both high and medium vigour embryos compared with the changes observed at 20°C, the differences being minor in the case of high vigour embryos but major for medium vigour embryos (Fig. 1B). At 10°C, high vigour
embryo polyA ÷ RNA levels decrease much less during the first hour of germination than at 20°C but exhibit the same general trend of a decline in polyA÷ RNA content followed by an increase back to the original level found in quiescent embryos over 0--7 h of germination. In medium vigour embryos germinated at 10°C the level of polyA÷ RNA i n t h e embryo remains unchanged for up to 3 h of germination before declining to a level of about half that found in high vigour quiescent embryos. This level then changes very little up to 24 h of germination (Fig. 1B). As the total a m o u n t of extractable RNA is similar in high and reduced vigour embryos over the time period studied the relative changes in levels of polyA÷ RNA are a true indication of the differences in polyA÷ RNA contents as a proportion of total cellular RNA and can be assumed to reflect levels of presumptive messenger RNA present in these embryos at the different imbibition stages. Inhibition of polyA÷ RNA synthesis in wheat embryos during germination in the presence of 12 pM a-amanitin permits a study of the disappearance of stored polyA÷ RNA (stored potential mRNA) species during germination. The results of these studies are presented in Fig. 2. During germination of high and medium vigour embryos at 20°C in the presence of a-amanitin the disappearance of stored polyA÷ RNA in these embryos follows similar patterns {Fig. 2A,C). An initial sharp decline in polyA÷ RNA levels over the 0- to 1-h germination period to reach a level of polyA÷ RNA which is approximately 40% of that found in the quiescent embryo reflects the steep decline in polyA÷ RNA content observed during germination at 20°C in the absence of a-amanitin (Fig. 1A). However, upon continuation of germination in the presence of ~amanitin beyond I h, no subsequent increase in polyA÷ RNA levels is observed (Fig. 2A,C} and the level of polyA÷ RNA in high and medium vigour embryos continues to decline up to 8 h of germination at a significantly reduced rate compared with the decline in the first hour of germination. After this time little
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Fig. 1. Embryos were germinated for the required time at 20°C (A) or 10°C (B) and total cellular RN A extracted as described. PolyA÷ R N A content was estimated as described in the Materials and methods section. Results represent the average +_ S.E.M. of 6 independent determinations. The 100% level of c.p.m, bound, i.e. the quiescent high vigour embryo value, is equivalent to 96 ng poly A ÷ R N A / 1 0 ug total RNA sample. The remaining time points are expressed as a percentage of c.p.m, bound by R N A from quiescent embryos high vigour (I I); medium vigour ( I - - - - - - I ) .
p o l y A÷ R N A remains in the ~-amanitin-treated embryos. Slight differences in the rate of decay o f polyA÷ R N A in high and medium vigour embryos at 20°C are reflected in the half-life of the decay of polyA÷ RNA, this being 0.35 h in high vigour embryos but 1.2 h in medium vigour embryos. Upon germination at 10°C in the presence of 12 ~M ~-amanitin distinct differences are seen in the decay profiles of stored polyA÷ R N A in high and medium vigour embryos (Fig. 2B,D). The degradation of polyA÷ R N A in high vigour embryos commences without any apparent delay although the decay of polyA÷ R N A at 10°C is slightly slower, with a half-life of 1.6 h compared with 0.35 h at 20°C. Again, there is little polyA÷ R N A de-
tectable in high vigour embryos after 6--8 h of germination. However, in medium vigour embryos there is a pronounced delay before any significant reduction in the level of p o l y A÷ R N A in these embryos is observed (Fig. 2B,D) and only after 2 h of germination does the level of p o l y A÷ R N A in medium vigour embryos decline significantly reaching levels that are barely detectable by 8 h of germination. In contrast during the initial 2 h germination period the level of p o l y A ÷ RNA in high vigour embryos germinated in the presence of ~-amanitin falls by 50%. This initial delay period in polyA ÷ RNA decay in the presence of ~-amanitin is also seen in medium vigour embryos germinated at 10°C in the absence of ~-amanitin (Fig. 1B).
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HOURS OF IMBIBtTION Fig. 2. S t o r e d m R N A - d i r e c t e d p r o t e i n s y n t h e s i s in high a n d m e d i u m vigour w h e a t e m b r y o s . E m b r y o s were pulse labelled for 1 h at t h e t i m e s i n d i c a t e d using [ 1 4 C ] a m i n o acid m i x t u r e in t h e p r e s e n c e or a b s e n c e o f 12 # M aa m a n i t i n . T h e u p t a k e a n d i n c o r p o r a t i o n o f [ 1 4 C ] a m i n o acids was d e t e r m i n e d as d e s c r i b e d in t h e text. The results are p r e s e n t e d as a h i s t o g r a m a n d r e p r e s e n t t h e average + S.E.M. o f 3 i n d e p e n d e n t e x p e r i m e n t s . The ' 1 0 0 % level' o f [ '4C 1 a m i n o acid i n c o r p o r a t i o n is t a k e n as t h e level o f i n c o r p o r a t i o n o f [ '4C] a m i n o acids i n t o acid-precipitable m a t e r i a l in t h e 0--1-h pulse p e r i o d a f t e r c o r r e c t i o n for a n y d i f f e r e n c e s in u p t a k e o f r a d i o a c t i v e a m i n o acids by the e m b r y o s u n d e r d i f f e r e n t g e r m i n a t i o n c o n d i t i o n s [ 7 ] a n d was e q u a l to 1025 +_ 10 c.p.m, i n c o r p o r a t e d i n t o acidp r e c i p i t a b l e m a t e r i a l p e r e m b r y o . This value was t h e s a m e w h e t h e r a - a m a n i t i n was p r e s e n t or a b s e n t from t h e germ i n a t i o n m e d i u m d u r i n g t h e initial 0--1-h pulse period. T h e curves (-- - - --) r e p r e s e n t the d e c a y o f s t o r e d p o l y A + R N A species in w h e a t e m b r y o s d u r i n g g e r m i n a t i o n in t h e p r e s e n c e o f 12 # M ~ - a m a n i t i n at 20°C o r 10°C. P o l y A ÷ R N A levels were d e t e r m i n e d a n d e x p r e s s e d as d e s c r i b e d in t h e legend to Fig. 1.
Stored messenger I~NA and protein biosynthesis When e m b r y o s are germinated at 20°C in the absence of a-amanitin their in vivo rate of protein biosynthesis increases over the initial 7-h germination period [7]. During this period e m b r y o s of high vigour synthesise protein at higher rates than medium vigour embryos. However, b y 17 to 18 h of germination at 20°C high and medium vigour embryos synthesise protein at similar rates [7]. Germination of embryos at a stress germination temperature (10°C) results in a much lower rate of protein biosynthesis in both high and medium vigour embryos than is observed at 20°C. In addition high vigour embryos synthesise protein at higher rates than medium
vigour embryos throughout the initial 18-h germination period at 10°C [7]. Although the rapid increase in protein synthetic rate during germination is not seen in embryos germinated in ~-amanitin (Fig. 2), the rate of protein synthesis in these embryos does not reflect the falling level of polyA÷ RNA {potential m R N A ) species within the embryos. Upon germination of high vigour embryos at 20°C in the presence of~-amanitin the level of polyA÷ R N A in the embryo falls rapidly yet it is not until approx. 80% of the polyA÷ R N A found in quiescent high vigour embryos has been degraded that any significant decrease in protein biosynthetic rate in the embryos is observed {Fig. 2A). A similar situation is seen in embryos of medium vigour ger-
77 minated at 20°C. Although the initial rates of protein synthesis are lower in these embryos than in high vigour embryos, no reduction in in vivo protein synthetic rates is seen in medium vigour embryos, even though polyA÷ R N A levels fall dramatically over the first 3 h of germination, until approx. 70--80% of the polyA÷ R N A found in quiescent embryos of medium vigour has been degraded (Fig. 2C). After this time (2--3 h of germination) the rate of protein synthesis in medium vigour embryos decreases sharply. Upon germination of embryos at 10°C in the presence of ~-amanitin, high vigour embryos exhibit a short lag period {0--1 h) when protein synthesis occurs at a very low rate {Fig. 2B} before synthesising protein at 60--70% of the rate found in high vigour embryos at 20°C between 1--3 h of germination. During this period polyA+ R N A levels in the e m b r y o fall to 30% of the level found in quiescent high vigour embryos. It is only when the level of polyA÷ R N A falls below this level, i.e. from 3 h of germination onwards, than any significant decrease in the rate of protein biosynthesis is observed {Fig. 2B). When medium vigour embryos are germinated in the presence of ~-amanitin at 10°C an initial lag period (0 -1 h) during which protein synthesis is barely detectable is followed by a 3-h period where little difference is noted in the protein synthetic capacity of the embryos even though the level of polyA+ R N A in these embryos declines to about 40% of the level in quisecent medium vigour embryos (Fig. 2D). After this time, however, the continuing slow decay of polyA÷ R N A levels in the e m b r y o s is accompanied by a significant decrease in the in vivo protein synthetic rate. Throughout the initial 4 h of germination at 10°C in the presence of a-amanitin the rate of protein synthesis in high vigour embryos is significantly greater than the level in medium vigour embryos. Discussion Quiescent wheat embryos contain polyA÷ RNA species which are degraded rapidly during
the initial stages of germination to be replaced b y polyA÷ R N A species which are produced during germination as a result of de novo R N A biosynthesis. The results presented here suggest that germinating wheat embryos can degrade up to 60--70% of this stored polyA÷ R N A before any significant reduction in the rate of in vivo protein biosynthesis directed b y this polyA÷ R N A becomes apparent {Fig. 2 ). This observation suggests that only 30--40% of this stored polyA÷ R N A is available to the protein synthesising apparatus of the embryo for translation into protein during early germination. It is possible that t w o different sub-populations of polyA÷ R N A species exist in the quiescent e m b r y o , one population being actively involved in the initiation of protein synthesis during seed germination whilst the other population may be merely 'carried over' from the ripening period and would comprise that proportion of the polyA÷ R N A population which disappears rapidly upon germination without affecting the in vivo rate of protein biosynthesis in the e m b r y o (Fig. 2). If this is the case, the germinating wheat embryo must possess a mechanism whereby it can distinguish those polyA÷ RNA species which are to be degraded during the first 2--3 h of germination from those species which are to be conserved to support protein biosynthesis during this time. The answer to h o w this distinction can be achieved may lie not in the polyA÷ R N A per se b u t in the nature of the proteins which are associated with the polyA÷ R N A since in the cell the polyA÷ R N A species complex with specific proteins to form messenger ribonucleoprotein {mRNP) particles. Control of the onset of protein biosynthesis during the early hours of germination of wheat embryos may be analogous to the situation which exists during early embryonic development following fertilisation of echinoderm and amphibian eggs [22]. In these developing e m b r y o s it has been suggested that the translation of maternal m R N A species, which was repressed in the unfertilised egg, is switched on after fertilisation by a mechanism as yet u n k n o w n b u t which
78 m a y involve a role for the proteins specifically associated with the mRNP particles [23]. Different classes o f m R N P particles can be isolated f r o m several o t h e r eukaryotic sources, b o t h animal [24] and plants [25]. In the germinating wheat e m b r y o different size classes o f native m R N P particles have been shown to exist [25] and the half-life of the template RNA species associated with these particles is o f the order o f 2 h at optimal germination t e m p er atu r es [26]. Thus, it is possible t hat some m R N P species in the quiescent wheat e m b r y o are pre-destined for rapid degradation during early germination whilst o t h e r species (30--40% o f the population) are actively involved in the initiation of protein synthesis during germination, their fate being controlled, at least partially, by the nature o f the specific protein c o m p o n e n t s associated with t he different m R N P particles. Loss o f vigour in wheat seed can be correlated with p o ly A÷ RNA levels in the e m b r y o and the rate of protein biosynthesis in these e m b r y o s after 24 h of germination at 10°C [12,13]. Indeed the rate of in vivo protein biosynthesis in the e m b r y o at this time is indicative o f the vigour rating of the seed lot, whereas this correlation is not seen between rates of total RNA synthesis and the vigour o f t h e e m b r y o s [13]. A detailed study of the stored m R N A c o n t e n t of e m br yos f r om two o f t h e seed lots used in previous studies on loss o f vigour [13] is r e p o r t e d here and indicates t h a t a fraction of this stored m R N A is capable o f supporting protein synthesis in the e m b r y o during the period immediately following reh y d r a t i o n o f the embryos, even in the absence of de novo p o ly A÷ RNA synthesis, Loss of vigour o f a seed lot in the absence of any significant loss of viability is associated with a change in the p atte r n of decay o f stored messenger RNA in reduced vigour e m br yos germinated under sub-optimal conditions (Fig. 2B,D). However, little difference is seen in p o l y A÷ RNA decay patterns in high and m e d i u m vigour e m b r y o s germinated at 20°C, a result which correlates with observations demonstrating th at vigour differences between
seed lots are not observable under optimal germination conditions [2,4]. The molecular basis for these differences in polyA÷ RNA metabolism in seed lots of varying vigour during germination at sub-optimal temperatures is not know n but current investigations in this l a b o r a t o r y are aimed at understanding the factors controlling translatability, conservation and decay of potential m R N A species during germination and the importance of the lesions appearing in m R N A turnover during loss of vigour, and subsequently loss of viability in cereals. Acknowledgements The financial support of the Lord Rank Prize Funds is gratefully acknowledged. References 1 E.H. Roberts, Viability of seeds, E.H. Roberts (Ed.), Chapman and Hall Ltd. 1972. 2 D.A. Stormonth, Developments in the business and practice of cereal seed trading and technology. pp. 49--76, P.R. Hayward (Ed.), The Gavin Press, London, 1978. 3 D.A. Stormonth and D. Doling, Arable Farming, 6 (1979) 42. 4 J.G. Hampton, N.Z.J. Exp. Agric., 9 (1981) 191. 5 S. Sen and D.J. Osborne, Biochem. J. 166 (1977) 33. 6 C.M. Bray, Recent advances in the biochemistry of cereals, D.L. Laidman and R.G. WymJones (Eds.), Academic Press, New York, 1979, pp 147--173. 7 L.E. Blowers, D.A. Stormonth and C.M. Bray, Planta, 150 (1980) 19. 8 W.J. Peumans and A.R. Carlier, Biochem. Biophys. Pflanz., 176 (1981) 384. 9 S.S. Abu-Shakra and T.M. Ching, Crop Sci., 7 (1967) 115. 10 T.M. Ching, Plant Physiol., 51 (1973) 400. 11 S.A. Standard, D. Perret and C.M. Bray, J. Exp. Bot., 34 (1983) 1047. 12 C.A.D. Smith and C.M. Bray, Planta, 176 (1982) 413. 13 C.A.D. Smith and C.M. Bray, Plant Sci. Letts., 34 (1984) 335. 14 S. Spiegel and A. Marcus, Nature, 256 (1975)228. 15 M.E. Gordon and P.I. Payne, Planta, 130 (1976) 269. 16 P.I. Payne, Biol. Rev., 51 {1976) 329.
79 17 S. Spiegel, R.L. Obendorf and A. Marcus, Plant Physiol., 56 (1975) 502. 18 F.B. Johnston and H. Stern, Nature, 74 {1957) 170. 19 G. Brawerman, Methods Enzymol., 30 (1974) 605. 20 M. Aviv and P. Leder, Proc. Natl. Acad. Sci. (Wash.) 69 (1972) 1408. 21 M. Rosbash and D.J. Ford, J. Mol. Biol., 85 (1974) 87. 22 A. Marcus, S. Spiegel and J.D. Brooker, Control Mechanisms in Development, R.H. Meints and E.
23 24 25 26
Davies (Eds.), Plenum Press, New York and London, 1975, pp. 1--20. J.D. Richter and L.D. Smith, Nature, 309 (1984) 378. H.P. Schmidt, K. Kohler and B. Setyono, Mol. Biol. Rep., 9 (1983) 87. W.J. Peumans and A.R. Carlier, Planta, 136 (1977) 195. L.I. Caers, W.J. Peumans and A.R. Carlie~', Pianta, 144 (1979) 491.