Embryonal Dormancy and Photosynthetic Carbon Metabolism in Apple Seedlings

Institute of Botany, University of Warsaw, Poland

Embryonal Dormancy and Photosynthetic Carbon Metabolism in Apple Seedlingsl) URSZULA MACIEJEWSKA and S. MALESZEWSKI With 4 figures Received January 28, 1976 . Accepted April 2, 1976

Summary Photosynthetic 14C0 2 assimilation was compared in seedlings from stratified and dormant embryos in the early phase of their development. The kinetics of HC incorporation into the characteristic metabolites of Calvin cycle, into the C 4 -dicarboxylic acids and into the metabolites of the glycolic acid pathway was studied. Dormancy was found to affect the pathways of photosynthetic carbon assimilation. The flow of carbon assimilated by C 4-dicarboxylic acids and by glycolic acid pathway metabolites was found to be enhanced in seedlings grown from dormant embryos when compared with those grown from stratified ones.

Key words: dormancy, photosynthesis, Malus domestica

Introduction Apple seed dormancy is terminated when the seeds are treated with low temperatures in humid surroundings for a few months (cold stratification). Changes in the activity of some enzymes and in the level of some growth regulators during stratification have been noted (RUNDNICKI, 1969; SINSKA and LEWAK, 1970; LEWAK et aI., 1975). Structural changes in the cells of the embryonic axis were also observed beetwen the 20th and 60th day of stratification (KOPAN, 1972). Germination of non-stratified seeds is also possible when the seed coat and endosperm are removed. Embryos isolated from such dormant seeds are in the state of so-called embryonal dormancy, which is characterised by slower germination and by the occurence of other developmental anomalies (different development of cotyledons, elongation of hypocotyle, dwarfism: FLEMION, 1956; VEGIS, 1964; COME, 1967). 1) This work was partially suported by Polish Academy of Sciences, Contract No 09.1.7. Abbreviations: PGA, 3-phosphoglyceric acid; PEP, phosphoenolpyruvate; ABA, abscisic acid.

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It has been stated that the delay in the appearence of photosynthetic activity caused by embryonal dormancy - is more pronounced than the retardation of morphological development. This evidence suggested that the factor(s) responsible for embryonal dormancy influence(s) the development of seedlings and the appearence of photosynthetic apparatus in particular manner (MACIEJEWSKA et al., 1974). Recently, considerable differences in the activities of some photosynthetic enzymes have been shown between seedlings developing from dormant and from stratified seeds (RYe and LEWAK, 1975). This fact suggests that embryonal dormancy also affects the pathway of photosynthetic carbon metabolism. Therefore - to elucidate this problem - the kinetics of 14C incorporation into the products of photosynthesis after various periods of growth of embryos isolated from dormant and from stratified seeds were investigated.

Material and Methods Apple seeds (Malus domestica BORB. cv. ANToNovKA, 1973 collection) were obtained from the Seed Center Experimental Station (ZNOS) at Kur6w. Embryos were isolated (removal of seed coat and endosperm) from non-stratified seeds and from seeds after 90 days cold stratification. The conditions of stratification have been described earlier (LEw AK and SMOLENSKA 1968). The culture was carried out in lots of 30 embryos in Petri dishes (diameter 10 cm) on filter paper moistened with 5 ml of distilled water. The conditions were as follows: 12 hour day, light intensity 10 4 lux, day temperature 24 °C, night 20 dc. The culture of embryos from stratified seeds was continued for 6 days, the embryos isolated from dormant seeds - because of their slower development - were cultured for 16 days. Embryos at similar stage of development on the particular day of culture were used for each experiment. To characterise the rate of development of photosynthetic activity in the tissues of stratified and dormant embryos, measurements of 14C02 assimilation were carried out each day throughout the period of culture of the embryos. The 14C02 exposition time was 10 min. These measurements provided a basis for the choice of culture periods in which the kinetics of t4C incorporation into photosynthetic products could be compared in the tissues of stratified and dormant embryos. In the experiments the radioactivity of photosynthesis products was assayed after 1, 2, 5 and 10 min of 14C02 assimilation. 14C02 was supplied to plants placed in a plexiglass photosynthesis chamber (18 X 12 X 3 cm) integrated into a closed circulation system having a capacity of 4 X 10 3 cm 3 • The embryos were first illuminated for 5 min with 2 X 104 lux and then 14C0 2 was introduced into the system: the initial CO 2 concentration was 0.03 0/0. The total 14C0 2 radioactivity was 50 flCi, the rate of air flow was 4 Umin and the temperature inside the chamber was 24-25 dc. Immediately after exposure to 14C0 2 the plant material was killed with 80 Ofo boiling ethanol. The embryos were then homogenised and the soluble photosynthesis products were extracted with boiling 80 Ofo ethanol. The radioactive compounds were separated and analysed by ion-exchange chromatography, two-dimensional paper chromatography and radioautography (MALESZEWSKI and LEWANTY, 1972). The chromatogram fragments containing the radioactive products were eluted with water. The HC incorporated into starch was assayed after amylolytic hydrolysis of the ethanol non-soluble fraction. The radioactivity of all fractions and separated compounds was determined by liquid scintillation counting. Two series of experiments were carried out, each in triplicate.

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Results and Discussion

In the tissue of embryos isolated from stratified seeds photosynthetic act1Vlty, expressed as 14C02 assimilation in light, had already appeared after 2 days of culture and increased rapidly, a 10-fold increase being observed in the intensity of 14C02 assimilation between the 3rd and 6th day of culture. In embryos which had not been submitted to stratification a delay period of about 10 days was observed, during which the 14C02 assimilation increased very slowly. A rapid development of the

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Dormancy and photosynthesis in apple seedlings

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photosynthetic activity in non-stratified embryos first took place after 13 days of culture. The rate of the subsequent development was the same as in the stratified embryos (Fig. 1). These results are in agreement with those from our previous studies (MACIEJEWSKA et aI., 1974). The periods of culture for the comparison of the pathways of photosynthetic 14C02 assimilation in the stratified and dormant embryos were chosen according to the data presented in Fig. 1. The stage of morphological development of these embryos in the respective periods (A, Band C corresponding respectively to A', B' and C' in Fig. 1) was similar and so were the 14C02 assimilation rates (Fig. 2 a: Total). For comparison of the metabolism of assimilated 14C in the tissues of stratified and dormant embryos the changes in the radioactivity of particular products of photosynthesis were analysed (Fig. 2 b, c). Special attention was drawn to the characteristic metabolites of the Calvin cycle (PGA, sugar phosphates), metabolites of the glycolic acid pathway (glycolic acid, glycine and serine) and to the C 4-dicarboxylic acids (malic and aspartic acids). The rate of development of the particular pathway has also been characterised by the ratio of the increase in the 14C incorporation rate into pathwayspecific metabolites to the increase of the total photosynthetic activity in the selected periods of seedlings development (Table 1). After 1 and 2 min of 14C02 assimilation the PGA + sugar phosphates fraction

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and aspartic acids increased much more slowly in embryos from non-stratified seeds than did the total 14C02 assimilation during the development of the seedlings. In the stratified embryos the enhancement of the 14C incorporation rate into this fraction was more than 1.7-fold greater than the increase in total photosynthetic activity (Table1). The involvement of glycolic acid pathway intermediates in metabolism of carbon assimilated was evaluated by investigating the 14C incorporation into glycolic acid and into glycine and serine. In the youngest embryos from non-stratified seeds (N) the radioactivity of these products - expressed as a percentage of the total amount of 14C incorporated - was twice as high as in the stratified embryos. In the older seedlings (B', C') the increased contribution of these metabolites to the CO 2 assimilation process is also apparent but only after brief exposure to 14C02 (Fig. 2 b). The increase of incorporation of 14C into glycolic acid, glycine and serine was more rapid in both groups of embryos throughout the entire period of culture than was the increase in the total assimilation. This difference, however, was most pronounced in the stratified embryos (Table 1). Embryonal dormancy also brought about a decrease in the accumulation of assimilated carbon in sucrose. This was marked in the experiments in which the oldest seedlings were exposed to 14C02 for 10 min (Fig. 2 c). The decreased incorporation of 14C into sucrose was accompanied by an increased level of radioactivity in sugar phosphates, glucose and starch (Fig. 3). The technique applied did not allow any exact separation of PGA from the sugar phosphates. However one could roughly say that after 10 min of exposure to 14C02 the sugar phosphates contained about 90 per cent of the radioactive carbon of this fraction. These results suggest that the factors responsible for embryonal dormancy interfere with the synthesis of sucrose from sugar phosphates.

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Neither in the stratified nor in the non-stratified embryos did analysis of 14C02 assimilation products reveal radioactive free fructose irrespective of the period of exposure to 14C02. However, acid hydrolysis of labelled sucrose yielded equal amounts of radioactive glucose and fructose. The state of dormancy had no significant effect on the incorporation of 14C into the other soluble products of photosynthesis, which contained altogether about 10 0/0 of the total amount of carbon incorporated. The distribution of 14C in the photosynthesis products after 1 min exposition of the seedling to 14C02 also indicates that dormancy affects the pathways of assimilated carbon (Fig. 4). When 14C incorporation into PGA and sugar phosphates is used as an indicator of carbon flow through the C s pathway, it may be stated that embryonal dormancy causes the delay in the development of this pathway. The incorporation of 14C into the C s metabolites was less in the nonstratified embryos than in the stratified ones throughout the whole duration of culture. On the other hand, incorporation of 14C into metabolites of the glycolic acid pathway was enhanced in non-stratified embryos during the whole duration of their growth. Enhanced radioactivity of Crdicarboxylic acids was also noted in the youngest seedlings developing from dormant embryos (A'), but during their furhter development (B', C') the incorporation of 14C into malic and aspartic acids decreases.

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Changes in PEP-carboxylase activity in apple embryos from stratified and dormant seeds were found to be dependent on the stage of dormancy and on the duration of embryo culture (RYe and LEWAK, 1975). The activity of PEP-carboxylase in non-stratified embryos between the 6th and 16th days of culture (N-C') was about twice as high as in the stratified embryos in the corresponding period of development. These data are concordant with our conclusion that the involvement of C-dicarboxylic acids in the metabolism of assimilated carbon is larger in non-stratified than in stratified embryos. The differences in the metabolism of assimilated CO 2 between seedlings from stratified and dormant seeds indicate that the factors responsible for embryonal dormancy affect the photosynthetic pathways of carbon. Their effect is most pronounced during the first 6 days of embryo culture. The disappearance of these factors during the further culture of non-stratified seedlings, expressed by a rapid increase of the photosynthetic activity of these seedlings after 11 days, is related to the reduction of 14C incorporation into C 4 metabolites and to the increase of 14C in the PGA + sugar phosphates fraction. The differences observed in the incorporation of HC into final products of photosynthesis may be due to less effective translocation of photosynthetic products in tissues of seedlings from dormant seeds. This may result in enhanced 14C incorporation into products accumulated in the chloroplasts (starch). Embryonal dormancy of apple seeds was found by some authors to be related to the ABA level (PIENIAZEK and GROCHOWSKA, 1967; RUDNICKI, 1969). The effect of ABA on the photosynthetic apparatus was also demonstrated in several papers (WELLBURN et aI., 1973; SANKHLA and HUBER, 1974). The decrease in the 14C02 assimilation rate and in the RuDP carboxylase activity was stated by SANKHLA and HUBER (1975), who grew wheat seedlings in the presence of ABA. A simultaneous increase in PEP carboxylase activity was also reported, and 10 sec of 14COz assimilation resulted in a higher radioactivity in malate and a lower in PGA. This evidence provides grounds for the consideration of ABA as a possible factor responsible for the different courses of development of photosynthetic activity in stratified and dormant embryos. Acknowledgements The autors thank Prof. dr. ST. LEWAK for his valuable suggestions and critical reading of the manuscript.

References COME, D.: L'inhibition de germination des graines de Pommier Pirus malus L. non dormantes. Role possible des phenols tegumentaires. Ann. Sc. nat. bot. 8, 371-378 (1967). FLEMION, F. : Effects of temperature, light and nutrients on physiological dwarfing in Peach seedlings. Plant Physiol. 31, 413-439 (1956).

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KOPAN, W. P.: Structural and biochemical changes in apple seeds during stratification (in Russian). Sel'skochozjajstviennaja biologija. 7, 126-128 (1972). LEWAK, ST., A. RYCHTER, and B. ZARSKA-MACIEJEWSKA: Metabolic aspects of embryonal dormancy in apple seeds. Physiol. Yeg. 13, 13-22 (1975). LEWAK, ST., and G. SMOLENSKA: Le systeme phytochrome dans la germination des semences de Pommier. Physiol. Veg. 6,403-408 (1968). MACIEJEWSKA, U., M. RYe, S. MALESZEWSKI, and ST., LEWAK: Embryonal dormancy and the development of photosynthetic activity in apple seedlings. Physiol. Veg. 12, 115-122 (1974). MALESZEWSKI, S., and Z. LEWANTY: Effect of oxygen concentration on the incorporation of 14C into products of photosynthesis in Balsam Fir. Z. Pflanzenphysiol. 67, 305-310 (1972). PIENIAZEK, J., and M. J. GROCHOWSKA: The role of natural growth inhibitor (Abscisin II) in apple seeds germination and the changes in content of phenolic substances during stratification. Acta. Soc. Bot. Polon. 36, 579-587 (1967). RUDNICKI, R.: Studies on abscisic acid in apple seeds. Planta. 86, 63-68 (1969). RYe, M., and St. LEWAK: Activity of phosphoenolpyruvate carboxylase in apple seedlings in relation to embryonal dormancy. Photosynthetica. 9, 299-303 (1975). SANKHLA, N., and W. HUBER: Enzyme activities in Pennisetum seedlings germination in the presence of abscisic and gibberellic acid. Phytochemistry. 13, 543-546 (1974). - - Regulation of balance between C a and C 4 pathway. Z. Pflanzenphysiol. 74, 267-271 (1975). SINSKA, I., and ST. LEWAK: Apple seeds gibberellins. Physiol. Yeg. 8, 661-667 (1970). WELLBURN, F. A. M., A. R. WELLBURN, J. L. STODART, and K. J. TREHARNE: Influence of gibberellic acid and absicisic acid and growth retardant CCC upon plastid development. Planta. 111, 337-346 (1973). VEGIS, A.: Dormancy in higher plants. Ann. Rev. Plant Physiol. 15, 185-224 (1964). U. MACIEJEWSKA, and S. MALESZEWSKI, Department of Plant Physiology and Isotope Laboratory, Institute of Botany, University of Warsaw, 00-927/1 Warsaw, ul. Krak.-Przedm. 26/28, Poland.

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