The Control of Food Mobilisation in Seeds of Cucumis sativus L. II. The Role of the Embryonic Axis H.V. Davies and J.M. C h a p m a n Department of Biology, Queen Elizabeth College (University of London), Campden Hill Road, London W.8, 7AH, U.K.
Abstract. Removal of the embryonic axis from germinated cucumber seeds, either on the second of fourth day after imbibition, subsequently results in reduced rates of lipid and protein degradation and in the accumulation of free sugars and amino acids in the cotyledons. These isolated cotyledons show an inherent capacity for expansion growth which apparently results from an increased rate of water uptake. When water uptake is inhibited by incubating samples in polyethylene glycol the rate of lipid degradation is further reduced. This is accompanied by an additional increase in the reducing sugar and sucrose content of the cotyledons. Protein degradation in isolated cotyledons is inhibited to the same extent whether samples are incubated in water or polyethylene glycol. Furthermore, amino acid levels show appreciable and almost identical increases in both incubation media. Evidently an inverse correlation exists between rates of reserve mobilisation and levels of end products. It is suggested that the axis controls food mobilisation through a sink effect by reducing the levels of end products in the cotyledons.
(Guardiola and Sutcliffe, 1971). Such observations result from the fact that end products of the mobilisation process are translocated away from the storage organs to meet both the energy and structural requirements of the growing axial tissues (Ching, 1966; Kriedemann and Beevers, 1967). Removal of the embryonic axis from seeds of Cucumis results in the inhibition of both lipid and protein degradation. Axis excision does not, however, inhibit the development of maximal activities of enzymes associated with the mobilisation of either type of food reserve (Slack et al., 1977; Becker et al., 1978; Davies and Chapman, 1979). Since a growing axis evidently provides an active sink for the soluble products of reserve mobilisation, this function of the embryonic axis has been investigated as a possible control mechanism for lipid and protein degradation in cucumber cotyledons. Materials and Methods Germination of Seeds
Key words: Cucumis
Seeds (food mobilisation).
Introduction The mobilisation of food reserves in seeds is known to follow germination and accompany growth of the embryo or embryonic axis. Lipid degradation, for example, appears to be correlated with axis growth in watermelon (Kagawa et al., 19"73) and Ponderosa pine (Ching, 1970), and protein breakdown with axis growth in squash (Wiley and Ashton, 1967) and pea Abbreviation: PEG=polyethylene glycol
Seeds of Cucumis sativus L. cv. Ridge Long Green were thoroughly washed in distilled water and imbibed for 3 h. Samples from the experimental treatment employed i.e. without testa or without testa and embryonic axis, were incubated in seed trays at 25+ 1~ C in the dark as described by Slack et al. (1977). For petri dish experiments 10 pairs of isolated cotyledonswere incubated in 9 cm diameter dishes containing two Whatman No. 1 filter paper discs and 5 cm3 of either distilled water or polyethylene glycol (PEG). A solution of PEG providing an osmotic potential of - 15 bar was prepared by the method of Slack (1978). Samples incubated in PEG were subsequently washed in distilled water prior to further analysis. Determination of Fresh Weight, Dry Weight, Protein and Lipid
Changes in fresh and dry weights were measured as described previously (Slack et al., 1977). The methods of Davies and Chap-
H.V. Davies and J.M. Chapman: Food Mobilisation in Seeds. II
"] cL d 0"10
Fig. 1a-c. Changes in the reducing sugar (a), sucrose (b) and amino acid (c) content of cucumber cotyledons with testas removed and axes present (o), and with both testas and axes removed (9 c.p., cotyledon pair
man (1979) were used to estimate total protein, total lipid, and changes in neutral lipid components.
Determination of Reducing Sugars, Sucrose and Free Amino Acids
Batches of 10 cotyledon pairs were homogenised in 5 cm3 of boiling 80% ethanol and the homogenates washed into 50 cma beakers with a further 30 cm3. Extraction in boiling ethanol was continued for 10 rain and samples centrifuged at 3,000 g for 5 rain. Supernatants were evaporated to dryness and lipids and pigments removed with 2.10 cna3 volumes of diethyl ether. Amino acids and sugars were then dissolved in 10 cm 3 of distilled water, Amino acid levels were determined by the method of Yemm and Cocking (1955) and total reducing sugars by the method of Nelson and Somogyi (Somogyi, 1952). Sucrose was determined as reducing sugar equivalents following the incubation of extracts with invertase (Bergmeyer, 1963).
sugars a n d free a m i n o acids appears to be inversely p r o p o r t i o n a l to the reduced rates o f lipid a n d p r o t e i n m o b i l i s a t i o n previously n o t e d for identically treated samples (Davies a n d C h a p m a n , 1979). S u b s e q u e n t rates of p r o t e i n a n d lipid d e g r a d a t i o n are also affected when the axis is r e m o v e d o n the second or f o u r t h day after i m b i b i t i o n a n d o n b o t h occasions a n appreciable i n h i b i t i o n is evident (Fig. 2).
C o m p a r e d with samples in which e m b r y o n i c axes are attached the excision of axes f r o m freshly i m b i b e d ( n o n - g e r m i n a t e d ) seeds results in a m a r k e d increase in the levels of reducing sugars a n d sucrose in the cotyledons (Fig. 1 a, b). Thus, c o m p a r e d with n o n excised material, sucrose levels in the isolated cotyledons are 2- a n d 3-fold higher, respectively, by the third a n d sixth day after i m b i b i t i o n . Similarly, a 3fold difference in the r e d u c i n g sugar c o n t e n t is s h o w n at day 6. Sucrose, however, constitutes the m a j o r c o m p o n e n t of the pool of soluble sugars in b o t h treatm e n t s t h r o u g h o u t the e x p e r i m e n t a l period, As Figure 1 c shows, a m i n o acid levels are also affected by axis excision a n d from the second day o n w a r d s rem a i n c o n s i d e r a b l y higher in isolated cotyledons t h a n in their attached counterparts. The a c c u m u l a t i o n o f
0 ~-8 O
2 ~ 6 8 days after imbibition
Fig. 2. The effect of axis excision on protein and lipid mobllisation in cucumber cotyledons. Axes were excised either on day 2 (---o---) or day 4 (--- 9 Axes were not removed in control experiments (o). Testas were removed at day 0
H.V. Davies and J.M. Chapman: Food Mobilisation in Seeds. II a
;/ "7 o: O'08d
-O: . . . .
Fig, 3. The effect of axis excision on levels of reducing sugars (a), sucrose (b) and amino acids (c) in Cucumis cotyIedons. Axes were either not removed (o) or excised either on day 2 (---o---) or day 4 (-- 9 - ). Testas were removed at day 0. c.p., cotyledon pair
y ./ /
.15 o. d
?e~. 8 0 .
E_ O~ "10 ~
days after irnbibition
days after imbibition
Fig. 4. Changes in cotyledon fresh weight (solid line) and dry weight (broken line) in the presence (e) and absence (o) of the embryonic axis. Testas were removed at day 0. c.p., cotyledon pair.
Fig. 5. The effect of incubating cotyledons from 4d-old seedlings in either PEG (o) or distilled water (e) on subsequent changes in fresh (solid line) and dry (broken line) weights. The effect of transfering samples from PEG to distilled water is also shown (A). c.p., cotyledon pair
As with the removal of the axis from freshly imbibed seeds, excision either at day 2 or 4 results in the accumulation of higher reducing sugar levels in isolated cotyledons compared with cotyledons of the intact seedling. Removal of the axis at day 2 results in an appreciably higher reducing sugar content for isolated cotyledons only after a subsequent 4 d incubation period (Fig. 3a). In contrast, if axis excision is performed on day 4 substantially higher levels for this treatment are observed after only 24 h. With sucrose, however, excision of the axis on day 2 does result in appreciably higher levels for isolated cotyledons after only 24 h (Fig. 3 b). Nevertheless, the rate of accumulation in this period after excision is still higher when cotyledons are isolated at day 4. As Figure 3c demonstrates, cotyledons separated from their axes either at day 2 or 4 also accumulate higher levels
of free" amino acids. Thus 48 h after axis excision levels are 40% higher in isolated cotyledons than in their attached counterparts. It should be noted, however, that these differences are relatively small in comparison with those shown for reducing sugars and sucrose. In the seed tray experiments it became apparent that isolated cotyledons in direct contact with the water supply visibly expanded as the incubation period was extended. Furthermore, these samples showed an accelerated increase in fresh weight but a reduced rate of dry weight loss when compared with cotyledons of the intact seedling (Fig. 4). Probably due to a combination of the cut surface, the increased surface area in contact with t h e water and the increased solute levels in the early stages of incubation, isolated cotyledons absorb and retain more
H.V. Davies and J.M. Chapman: Food Mobilisation in Seeds. II
0LI . . . . d
r O~ =..
:,, . . . .
days after imbibition Fig. 6. The effect of incubating cotyledons from 4 d-old seedlings in either PEG (o) or distilled water ( e ) on protein and lipid degradation. The effect of transfering samples from PEG to distilled water is also shown (--- 9 ---)
En E 0.2-
"T 0.2 d. ,j
E 0.1 0"2'
8 12 imbibition
Fig. 7a-d. Changes in the relative levels of triglycerides (a), 1,2 diglycerides (b), 1.3 diglycerides (e) and free fatty acids (d) in cotyledons from 4 d-old seedlings incubated in either PEG (9 or distilled water ( e )
water. This enhanced uptake of water evidently facilitates cotyledon expansion and the possiblility that such expansion may be related to food mobilisation was then examined. Figure 5 illustrates the effect of incubating excised cotyledons in petri dishes, containing either distilled water or PEG, on subsequent changes in fresh and dry weights. Cotyledons were excised from 4 d-old seedlings at which time the enzymes associated with either lipid or protein mobilisation showed maximal activities (Davies and Chapman, 1979). Incubation in PEG inhibits both the normal increase in fresh weight and the decrease in dry weight shown by cotyledons incubated in water alone. This treatment therefore
4 8 12 4 8 days after imbibition
Fig. 8. Changes in the levels of amino acids (a), reducing sugars (b) and sucrose (c) in cotyledons from 4 d-old seedlings incubated in either PEG (o) or distilled water ( e ) e.p., cotyledon pair '
inhibits water uptake and, as would be expected, also visibly suppresses cotyledon expansion. In addition, samples incubated in PEG show reduced rates of lipid degradation although this effect, together with the inhibition of water uptake (and consequently cotyledon expansion), is a reversible one (Fig. 6). The influence of PEG on total lipid levels clearly reflects the reduced rates of triglyceride, diglyceride and free fatty acid utilisation that result from this treatment (Fig. 7). Protein degradation is almost totally inhibited in the period following axis excision, both when cotyledons are incubated in water and PEG (Fig. 6). In contrast, amino acid levels increase in both cases,
H.V. Davies and J.M. Chapman: Food Mobilisation in Seeds. II
although there is never a substantial difference between treatments (Fig. 8a). In contrast, although both PEG and water treatments produce an increase in the levels of reducing sugars and sucrose in the cotyledons the increase in the PEG treatment remains higher throughout the 7d period subsequent to axis excision (Fig. 8b, c). As far as both lipid and protein degradation are concerned, therefore, there is a clear correlation between the inhibition of hydrolysis and the accumulation of products associated with the mobilisation of these polymeric reserves.
Excision of the embryonic axis from freshly imbibed seeds of Cucumis results in substantially reduced rates of lipid and protein mobilisation in the cotyledons (Slack et al., 1977; Davies and Chapman, 1979). In the related species, Cucurbita maxima, such an effect has been correlated with an axial requirement for the development of optimal activities of enzymes associated with the mobilisation of either type of food reserve (Penner and Ashton 1967b; Tsay and Ashton, 1974). It has been suggested that hormones transported from the axis are involved in the control of such enzyme activity (Penner and Ashton, 1967a, b; Sze and Ashton, 1971). However, experiments with cucumber cotyledons have failed to demonstrate that the presence of the axis is required for the normal development of enzyme activity (Slack et al., 1977; Davies and Chapman, 1979). Thus if a hormonal mechanism is operative it must influence in vivo enzyme activity. The present investigation has demonstrated that following the removal of the axis an inverse correlation exists between the subsequent inhibition of lipid and protein hydrolysis and the accumulation of sugars and amino acids in the cotyledons. This correlation exists irrespective of the time of axis excision. This suggests that the axis could exert its influence on reserve mobilisation by removing the soluble end products of lipid and protein degradation via a sink effect. By this mechanism the amino acid and sugar content of the cotyledons may be held below a critical level above which enzyme activity is suppressed and reserve mobilisation inhibited. Other examples exist in the literature which suggest that this mechanism operates in the control of food mobiIisation. The endosperm of castor bean, for example, develops lower activities of fl-oxidation, glyoxylate cycle and reverse glycolysis enzymes but accumulates higher levels of sugars when the cotyledons and embryos are excised (Huang and Beevers, 1974). In addition, the application of exogenous glucose partially suppresses isoci-
trate lyase activity in the endosperm (Lado et al., 1968). Glucose application also inhibits development of this enzyme in cotyledons of squash (Lado et al., 1968), and treatment of pea seeds with amino acids inhibits protease development in the cotyledons (Yomo and Varner, 1973). In Cucumis, however, the accumulation of sugars and amino acids is not associated with an inhibition of enzyme development. Thus in this system any proposed mechanism for the control of enzyme activity in vivo must consider the possibility of feedback phenomena. If the sink hypothesis holds true then the fact that some lipid degradation does occur in the absence of the axis suggests that cucumber cotyledons themselves possess a limited internal sink. The data obtained from experiments with PEG imply that the development of such a sink depends upon the inherent capacity of isolated cotyledons for expansion growth. When, therefore, expansion is prevented by restricting water uptake a further reduction in lipid mobilisation is observed which is again inversely correlated with an additional increase in sugar levels. Since cell expansion requires the synthesis of new cell wall material (Ray, 1962) the utilisation of accumulated sugars for this purpose may allow limited lipid mobilisation by maintaining the sugar content below completely inhibitory levels. The application of exogenous sucrose to isolated cotyledons also results in a further inhibition of lipid hydrolysis, but not through an osmotic effect (Slack et al., 1977). Hence by inducing an increase in sugar levels, either by restricting cotyledon expansion or by the application of an exogenous supply, lipid degradation can be inhibited in the storage organs. An inverse correlation also exists between the inhibition of protein degradation and the accumulation of amino acids in the cotyledons. There is, therefore, an indication that an axial sink may be required for optimal rates of protein mobilisation. However, if the sink hypothesis holds true for this reserve then the proteolytic enzymes must be sensitive to the relatively small increases in free amino acid levels that result from axis excision. Furthermore, almost identical increases in amino acid levels occur in expanding cotyledons and in samples where expansion is restricted. Since proteolysis is almost totally inhibited in both cases it appears that the cotyledon expansion process does not provide a sink sufficiently active to prevent the accumulation of inhibitory levels of amino acid. The suggestion that the embryo or embryonic axis controls food mobilisation via the production of a hormonal stimulus is usually based on the observation that exogenously applied hormones, cytokinins and gibberellins in particular, may partially or completely replace the axial requirement for normal enzyme de-
590 v e l o p m e n t ( B l a c k a n d Altschul, 1965; P e n n e r a n d A s h t o n , 1967a; Sze a n d A s h t o n , 1971). C y t o k i n i n a p p l i c a t i o n is k n o w n to result in a p r o m o t i o n of cotyl e d o n e x p a n s i o n ( M i k u l o v i c h e t a l . , 1971; L o n g o et al., 1978) a n d the same is true for gibberellins (Pinfield a n d Stobart, 1972). I n view of the c o r r e l a t i o n between lipid m o b i l i s a t i o n a n d c o t y l e d o n e x p a n s i o n in C u c u m i s our results show that care m u s t be t a k e n when i n t e r p r e t i n g results o b t a i n e d with h o r m o n e application studies. O u r data implies that the c o n t r o l of food m o b i l i s a t i o n in c u c u m b e r involves the m a i n tenance, via a n axial sink, o f n o n - i n h i b i t o r y levels of sugars a n d a m i n o acids in the cotyledons. As far as lipid m o b i l i s a t i o n is c o n c e r n e d p r e l i m i n a r y experim e n t s have been u n a b l e to d e m o n s t r a t e a n i n h i b i t o r y effect of either glucose or sucrose o n in vitro activities of lipase, isocitrate lyase a n d f r u c t o s e - l , 6 - b i s p h o s p h a tase. By r e m o v i n g the axial sink, therefore, the subseq u e n t a c c u m u l a t i o n of sugars m a y induce the sequential b u t partial suppression of i n d i v i d u a l reactions in the lipid-sugar c o n v e r s i o n p a t h w a y t h r o u g h end product inhibition phenomena. H.V. Davies acknowledgesthe receipt of an A.R.C. Research Assistantship.
References Becker, W.M., Leaver, C.J., Weir, E.M., Riezman, H. : Regulation of glyoxysomal enzymes during germination of cucumber 1. Developmental changes in cotyledonary protein, RNA, and enzyme activities during germination. Plant Physiol. 62, 542 549 (1978) Bergmeyer, H.-U. : Methods of enzymic analysis, pp. 92-102. New York, London: Academic Press 1963 Black, H.S., Altschul, A.M.: Gibberellic acid - induced lipase and c~-amylaseformation and their inhibitionby aflatoxin. Biochem. Biophys. Res. Commun. 19, 661-664 (1965) Ching, T.M. : Compositional changes of douglas fir seeds during germination. Plant Physiol. 41, 1313-1319 (1966) Ching, T.M.: Glyoxysomes in megagametophyte of germinating ponderosa pine seeds. Plant Physiol. 46, 475 482 (1970) Davies, H.V., Chapman, J.M.: The control of food mobilisation in seeds of Cucumis sativus L. I. The influence of embryonic axis and testa on protein and lipid degradation. Planta 146, 579 584 (1979)
H.V. Davies and J.M. Chapman: Food Mobilisation in Seeds. II Guardiola, J.L., Sutcliffe, J.F.: Control of protein hydrolysis in the cotyledons of germinating pea (Pisum sativum L.) seeds. Ann. Bot. 35, 791 807 (1971) Huang, A.H.C., Beevers, H. : Developmental changes in endosperm of germinating castor bean independent of the embryonic axis. Plant Physiol. 54, 277-279 (1974) Kagawa, T., McGregor, D.I., Beevers, H.: Development of enzymes in the cotyledons of watermelon seedlings.Plant Physiol. 51, 66-71 (1973) Kriedemann, P., Beevers, H.: Sugar uptake and translocation in the castor bean seedling 1. Characteristics of transfer in intact and excised seedlings. Plant Physiol. 42, 161 173 (1967) Lado, P., Schwendimann, M., Marre, E.: Repression of isocitrate lyase synthesis in seeds germinated in the presence of glucose. Biochem. Biophys. Acta 157, 140-148 (1968) Longo, G.P., Olginati, M., Rossi, G., Valente, M., Longo, C.P. : Effect of brief treatments with benzyladenine on growth and development of watermelon cotyledons. Plant, Cell and Environment 1, 39 43 (1978) Mikulovich, T.F., Khokhlova, V.A., Kulaeva, O.N., Sveshnikova, I.N. : Effect of 6-benzyl aminopurine on isolated pumpkin cotyledons. Fiziol. Rast. 18, 79 87 (1971) Penner, D., Ashton, F.M. : Hormonal control of proteinase activity in squash cotyledons. Plant Physiol. 42, 791 796 (1967a) Penner, D., Ashton, F.M.: Hormonal control of isocitrate lyase synthesis. Biochim. Biophys. Acta 148, 481485 (1967b) Pinfield, N.J., Stobart, A.K. : Hormonal regulation of germination and early seedling development in Acer pseudoplatanus (L.). Planta 104, 134-145 (1972) Ray, P.M. : Cell wall synthesis and cell elongation in oat coleoptile tissue. Am. J. Bot. 49, 928~39 (1962) Slack, P.T. : The control of lipid mobilisation in Cucumis cotyledons. Ph.D. thesis, Queen Elizabeth College, University of London 1978 Slack, P.T., Black, M., Chapman, J.M. : The control of lipid mobilisation in Cucumis cotyledons. J. Exp. Bot. 28, 569 577 (1977) Somogyi, M. : Notes on sugar determination. J. Biol. Chem. 195, 1943 (1952) Sze, H., Ashton, F.M.: Dipeptidase development in cotyledons of Cucurbita maxima during germination. Phytochemistry 10, 2935-2942 (1971) Tsay, R., Ashton, F.M.: De novo synthesis and hormonal regulation of a dipeptidase in Cucurbita maxima. Phytochemistry 13, 1759-1763 (1974) Wiley, L., Ashton, F.M.: Influence of embryonic axis on protein hydrolysis in cotyledons of Cucurbita maxima. Physiol. Plant. 20, 688-696 (1967) Yemm, E.W., Cocking, E.C.: The determination of amino-acids with ninhydrin. The Analyst 80, 209-213 (1955) Yomo, H., Varner, J.E.: Control of the formation of amylases and proteases in the cotyledons of germinating peas. Plant Physiol. 51, 708-713 (1973) Received 26 March; accepted 15 May 1979
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