Planta (Berl.) 124, 287--295 (1975) 9 by Springer-Verlag 1975
Effect of Light on Nucleic-acid Synthesis and Polyploidy Level in Elongating Epicotyl Cells of Pisum sativum P. Van Oostveldt and R. Van Parijs Laboratory of Biochemistry, Faculty of Agricultural Sciences, State University, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium Received 27 January; accepted 25 March 1975
Summary. The synthesis of DNA and RINA and the increase in dry matter were followed in the elongating cells of the epicotyl of peas (Pisum sativum L.) germinating in total darkness or in continuous light. The amounts of DNA and I%NA per epicotyl were estimated by chemical methods, the amount of DNA per cell was measured by histophotomctric techniques. The increase in DI~A, t%I~TAand dry matter in the epicotyl cells is much higher during germination in darkness than in light. During elongation in the dark most cortical epicotyl cells reach the 8C polyploidy level, in the light only the 4C polyploidy level is reached. A decrease in RNA synthesis is in agreement with a reduction in nuclear volume.
Introduction The metabolism of nucleic acids (NAs) during cell elongation is a widely studied problem of plant physiology. A causal relationship between DNA synthesis and cell elongation was studied especially in two ways. In one type of experiments, tissues were treated with a plant hormone, such as gibberellin, which promoted cell elongation, and the enhanced DNA synthesis was followed by [all] thymidine incorporation (e.g. Degani and Atsmon, 1970). Other workers used light as an external factor which inhibited cell elongation ; the reduced DNA synthesis was again followed by [aH]thymidine incorporation (e.g. Bopp, 1970). In either type of experiment the causal relationship between DI~A synthesis and cell elongation was studied by blocking DNA synthesis with 5-fluorodeoxyuridine and observing the inhibitory effect on cell elongation (Nitsan and Lang, 1965; Bopp, 1967a; Capesius et al., 1972). I n most cases cpicotyls or hypocotyls of germinating seeds were used. Most authors assumed t h a t the growth of epicotyl or hypocotyl occurs only by cell elongation without cell divisions. This implies t h a t DNA synthesis is restricted to extranuclcar DNA synthesis or to endomitotic DNA duplication. However, direct histophotometric estimations of the amount of DNA per cell, in order to determine the level of endomitosis, were rarely done (Capesius and StShr, 1974). Van Parijs (1967) followed the total amount of DNA, RNA, protein and dry matter during elongation in darkness of the epicotyl of Pisum sativum and established an endomitotic cell cycle (Van Parijs and Vandendriessche, 1966a). In this paper we report chemical determinations of Rb~A and DNA amounts in epicotyls of pea seedlings growing either in darkness or in light, together with histophotometric DNA measurements and deter-
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m i n a t i o n s of t h e n u c l e a r v o l u m e . I t w a s o u r i n t e n t i o n t o see w h e t h e r t h e i n h i b i t o r y e f f e c t of l i g h t o n cell e l o n g a t i o n is a c c o m p a n i e d b y a n i n h i b i t i o n of t h e t o t a l a m o u n t of N A s a n d b y a n i n h i b i t i o n i n t h e e n d o p o l y p l o i d y l e v e l of t h e e p i c o t y l ceils. Material and Methods
Plant Material. Seeds of the common garden pea (Pisum sativum L., cv. Rondo;SeedTrade Labor, Ghent, Belgium) were soaked in running tap water for 18 h. The epicotyls (internode between cotyledons and first leaf) were excised directly after soaking (0-day-old seedling) and after different periods of further germination. The seeds were germinated a t 15 ~ in plastic dishes filled with moistened polyester foam. A t each sampling, a b o u t 200 epicotyls were excised, collected in glass vials with dry ice, and stored in a freezer a t -- 15 ~ until use. For germination in continuous light the dishes were placed under a set of 10 fluorescent lamps ( " P h y t e r " ; ACEC, Charleroi, Belgium). The spectral energy distribution of these lamps is published in De Greef and Fredericq (1969). Light intensity at the seed level is 19 ~zW cm -2 n m at 650 n m a n d 7 ~W cm -2 n m at 450 nm. Extraction o/Nucleic Acids. The excised epicotyls were ground in a mortar with dry ice and further homogenized in a Teflon-glass Potter-E1vehjem homogenizer at 0 ~ After centrifugation (5000 • g for 10 min) the pellet was washed several times according to Ogur and l~osen (1950) to remove non-NA material. The NAs were extracted from the washed residue by heating in 5% trichloroacetic acid at 90 ~ for 15 min (Schneider, 1945). The total NA content of the extract was determined by U V absorption a t 268.5 n m (Logan et al., 1952) and b y phosphate determination after destruction of the NAs in 10 N HeSO 4 following the method of Burmaster (1946) as adapted by Steckx and Vandendriessche (1956). DNA was measured according to B u r t o n (1956) using deoxyribose in 5 % TCA as standard. R N A was calculated from the difference between the total ~ A content and the DNA content. The amounts of DNA and R N A were expressed as DNA-phosphorus (DNA-P) and RNAphosphorus (RNA-P). All measurements were done in quadruple. Dry Matter. A p a r t of the homogenate in 95 % ethanol was first dried a t low temperature, then at 100 ~ for 18 h. Histophotometric DNA Determination. Whole embryos, epicotyls or parts of the epicotyl were excised, fixed in A F A (ethanol 95?/o-formaldehyde 37%-glacial acetic acid, 75:20: 5, v/v) for 18 h at room temperature, dehydrated through tertiary butanol, and embedded in paraffin. Sections were cut a t 15 ~m for 0-day-old seedlings and at 25 [zm for 4- and 8day-old seedlings. Tissues of different stages were fixed, hydrolysed, and stained simultaneously in order to obtain comparable relative amounts of DNA per cell. Feulgen staining was applied after hydrolysis with 1N HC1 for 12 rain at 60 ~ The DNA content of the cortex nuclei was determined by the cytophotometric technique of Lison (1953, p. 139), using a multiple-plug measurement technique. I n this method a diaphragm is first directed beside the nucleus and a blank is read (transmission 100% ). Subsequently, different transmission readings (T) are t a k e n in the nucleus. The a m o u n t of DNA is expressed as log 1/T • area of the nucleus. The area of the nucleus is estimated b y planimetry. The transmissions were measured a t 556 n m (interference filter Filtraflex B-40; Balzer, Liechtenstein) in the nuclei of the epicotyl cortex, after 0, 4 and 8 days of germination. The plumule of the 4-day-old seedling (germination in the dark) was included as reference (diploid a m o u n t of DNA). Statistical Methods. Volumes were calculated from the formula of Puff (M6rike, 1953), i.e. V = 0.8488 F~/L wherein F ~ the area of the projected image of the nucleus, L = the length of the long axis. Regression lines were calculated on a programmed calculator (Diehl, Nfirnberg, Germany). Multimodal distributions of the a m o u n t of D•A per nucleus were separated in different unimodal populations by plotting the cumulative frequency diagram on probability paper according to the method of Cassie (1954). The percentages of the nuclei in the different populations were estimated b y considering the points of inflection of the curves (Rasch, 1970).
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Results
Chemical Determination o] Nucleic Acids and Dry Matter Nucleic-acid determinations were carried out from the 3rd day on. As shown by Van Parijs (1967) no synthesis of NA is occurring during the first 2 days. Starting with a net value of 0.23 ~g DNA-P per epicotyl a value of 0.82 and 0.62 9g DNA-P per epicotyl is reached in continuous dark and in continuous light, respectively (Fig. i). As can be seen in the figure this difference is highly significant. Thus light inhibits DNA synthesis. If the growth of the epicotyl is occurring by cell enlargement only, then we can assume that light reduces endomitosis. The amount of total NA-phosphorus per epicotyl reaches a m a x i m u m after 3 days, in the dark as well as in the light (Fig. 2). No significant differences are found between the total NA content as estimated from UV absorption (Logan et al., 1952) and from phosphorus determinations (Burmastcr, 1946). The ratio of RNA/DNA gives an indication for the amount of I~NA per cell (Fig. 3). This ratio reaches a maximum after 3 days of germination in the dark, but no clear m a x i m u m is found in the light. This difference between light- and dark-germinated seedlings is significant at the 5% level as was deduced from a non-parametric test. This test was used because the estimation of the standard deviation from a series of ratios is not appropriate. The test can be formulated as t--
Wl @ W2
wherein 21 and 2~ are the means of the two groups of ratios and wa and w~ are the ranges respectively of group 1 and group 2. The value t is compared with the value found in table (Crow et al., 1960). The I~NA/DNA ratio in the dark, after full elongation at 8 days, is nearly the same as the ratio I%NA/DNA in the light. The amount of dry matter of the epicotyl continues to increase at the time the amount of RNA per cell reaches a minimum. Finally the amount of dry matter in the light-grown epicotyl is about half the amount in the dark-grown epicotyl.
Histophotometric D N A Determinations in the Cortex o/ the Epicotyl 1. Polyploidy. The amounts of DNA, as measured by the Feulgen procedure, are shown in Table 1 and Fig. 4. As was to be expected from the results obtained with chemical methods the histophotometric determinations show t h a t most nuclei reach the oetaploid 8 C DNA level after 4 days of germination in the dark. After the same period of germination under continuous light, most of the nuclei have reached only the tetraploid DNA level. After 8 days of germination either in the dark or under continuous light, the DNA level per nucleus seems to be stabilized and there are no obvious differences between the situation after 4 days or after 8 days of germination. 2. Relation between D N A per Nucleus and Nuclear Volume. When the logarithm of the relative amount of DNA per nucleus is plotted against the logarithm of the nuclear volmne, a clear linear correlation is observed. From such plots (diagraphs) we calculated the relative nuclear volume corresponding to each
290
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Nucleic-acid Synthesis and Light
291
Table 1. Percentages of nuclei of different ploidy levels in cortex cells at different stages of epicotyl elongation Days, light conditions
2C
4C
8C
16C
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77
23 81 9O 14 19
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Fig. 1. Evolution of DNA-P/epicotyl in the dark (e) and in the light (o). Vertical bars = confidence interval at the 5 % probability level Fig. 2. Evolution of total nucleic acid-P/epicotyl in the dark (e) and in the light (o). Vertical bars ~ confidence interval at the 5 % probability level Fig. 3. I~NA-P/DNA-P ratio ( 9 o) and dry matter per epicotyl (A ~) under continuous dark (A e) and light (A o) Fig. 4. Histophotometric DNA determinations of individual nuclei. From top to bottom: plumule, after 4 days germination in the dark--epicotyl, before germination (0 days)--epicotyl, 4 days in continuous light~-epicotyl, 8 days in continuous light~-epicotyl, 4 days in continuous dark--epicotyl, 8 days in continuous dark
292
P. Van Oostveldt and R. Van Parijs
Table 2. Correlation analysis between the logarithm of the nuclear volume (y) and the logarithm of the amount of DNA (x) Relative nuclear volumes are calculated for 2C, 4C and 8C nuclei of the cortex of epieotyls germinating in the dark or in the light. Plumule nuclei are ehoosen as a reference Tissue, light conditionsa
Relative nuclear volume 2C
4C
8C
P1 4D Ep 4D Ep 8D Ep 0D Ep 4L Ep 8L
1 4.5 2.8 1.2 0.71 1.3
1.8 7.3 4.7 1.9 1.6 2.0
3.5 11.2 7.7 3.0 3.6 2.8
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0.90706 0.61937 0.74112 0.64194 1.11491 0.50344
0.10298 0.13170 0.07571 0.08288 1.14342 0.19007
0.66473 0.42908 0.62434 0.69560 0.66798 0.25847
100 100 152 66 77 100
8.81 4.70 9.79 7.74 7.77 2.65
a P1 = plumule, Ep = epicotyl ; 0 = 0 days, 4 = 4 days, 8 = 8 days; D = continuous darkness, L = continuous light. b by=+x = regression coefficient of y from x. e ay+x = standard deviation of by_+x . d r = correlation coefficient. e N = total amount of nuclei measured. f t = Student's t for the correlation coefficient.
polyploidy level in order to get some i n f o r m a t i o n a b o u t the a c t i v i t y of the nuclei. The results of these calculations are shown in Table 2. Since the diploid nuclei from 4-day-old dark-grown p l u m u l e s have the smallest volume, t h e y can be considered as physiologically less active (St6ekert, 1962). These nuclei were used as reference (volume = 1). I n the dark-grown tissues the nuclei, as deduced from their relative volume, seem to be more active t h a n in the light-grown tissue. 3. Changes, Other than D o u b l i n g s , i n the A m o u n t o / D N A per N u c l e u s . Earlier histophotometrie D N A d e t e r m i n a t i o n s have shown t h a t the diploid cortex cells of the epicoty] before g e r m i n a t i o n c o n t a i n a b o u t 20 % less D N A t h a n the diploid meristematie cells of the p l u m u l e (Van Parijs a n d Vandendriessehe, 1966b) a n d the same difference was f o u n d between 8 C nuclei of young, n o t fully elongated cortex cells a n d 8 C nuclei of fully elongated cortex cells. This m e a n s t h a t in the y o u n g 8C nuclei of cells which prepare for elongation, some D N A is missing b u t is present later when the cells are fully elongated. I n our results (Fig. 4) a n analogous difference is found at the 4C D N A level between the cortex cells of 4- a n d of 8-day-old epieotyls grown i n the light. The fact t h a t this difference is f o u n d during g e r m i n a t i o n in the light as well as i n the dark indicates t h a t influences of physical errors, such as d i s t r i b u t i o n a l error or the SehwartzschildVilliger effect (Sandritter, 1958) which are d e p e n d e n t on the e x t i n c t i o n values, are n o t very likely. The e x t i n c t i o n of the F e u l g e n stain (DNA per v o l u m e ; Table 2) in nuclei of light- a n d dark-grown cortex varies b y a factor 2 or 3. B u t the differences in the a m o u n t of F e n l g e n stain between nuclei at the b e g i n n i n g a n d those at the end of elongation are f o u n d in dark as well as in light. More evidence for the absence of some D N A fraction a n d indications a b o u t the n a t u r e of this D N A will be presented in a n o t h e r publication.
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Discussion Our results show t h a t inhibition of cell elongation by light in the epicotyl of Pisum sativum is accompanied by a reduction of endomitosis in the cortex cells. This process of endomitosis is completely finished after 4 days of germination and precedes growth in length of the epicotyl since elongation is closely parallel to dry-matter increase (van Parijs, 1967). The inhibition of cell elongation by light involves also a reduced synthesis of I~NA. In contrast to the behaviour of Lupinus albus hypoeotyls (Giles and Myers, 1964) we noted a sharp decrease in the total amount of RNA per epicotyl in Pisum sativum after 4 days in the dark. I t m a y be argued that the difference in the metabolism of RNA (especially synthesis of ribosomes) between Lupinus and Pisum is related to differences in the germination physiology. In Pisum the elongation of the epieotyl m a y be followed by the elongation of the higher internodes up to the sixth (Low, 1971). RNA synthesized in the epicotyl is supposed to break down and to be transported into the higher internodes. In Lupinus, the hypocotyl is the only internode which is growing in the dark; thus in this case RNA is needed during the whole germination process. This hypothesis is supported by the fact that transfer of Lupinus seedlings from dark to light causes a drastic decrease in the amount of RNA (Giles and Myers, 1964). The hydrolyzed RNA can then be used in the higher internodes which are elongating in the light. A number of investigators demonstrated a relationship between DNA synthesis and cell elongation (Nitsan and Lang, 1965; Bopp, 1967a, 1970; Capesius et al., 1972). However in most eases these studies did not include histophotometrie DNA determinations and therefore it was not possible to conclude if DNA synthesis was restricted to endomitosis. Only recently Capesius and St6hr (1974) have shown t h a t inhibition of cell elongation by 5-fluorodeoxyuridine is accompanied by a reduced endomitotie cell cycle. In germinating seeds the process of endomitotie DNA synthesis seems to occur very early. In Sinapis alba most DNA replication is finished 60 h after germination (Capesius and Bopp, 1970; Capesius and StShr, 1974). Light as an external factor can irreversibly block this DNA synthesis (Capesius and Bopp, 1970). I n our experiments DNA synthesis was slowed down by growing the seedlings at 15 ~ Nevertheless after 4 days, at the stage the epieotyls have reached half their final length, the endomitotic activity was finished. In older material (8-10 days) a number of cells are dividing mainly in the prevaseular tissue where adventious roots arise. Van Parijs and Vandendriessche (1967 a) carried out DNA measurements in the prevascular tissue and found t h a t these nuclei were blocked at a 4C DNA level after 4 to 8 days, probably corresponding to nuclei in a premitotic G 2 phase. From transverse sections of epicotyls (8-day-old) we found that about 2500 cortex cells and 1500 cells of the prevascular elements are present in the epicotyl. Epidermis cells were not counted. If we know the amount of DNA at 0 days, then we can calculate the total amount of DNA after 4 days germination in light or dark, assuming that all the 1500 prevascular cells become tetraploid and that the cortex cells reach the levels of polyploidy shown in Table 1. In this way we find t h a t in the light total amount of DNA
294
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will be 0.45 ~g. Experimentally, a value of 0.65~zg is found. For germination in the dark we calculate an a m o u n t of D N A of 0.68 ~g while experimentally a value of 0.80 ~zg is found. The calculated ratio of the a m o u n t D N A per epicotyl in the dark and the a m o u n t of D N A per epicotyl in the light, equals 1.50 while experimentally a ratio of 1.23 is found. A Z 2 test shows t h a t the calculated amounts of DNA are in agreement with the experimental results. The enhanced D N A and RIgA synthesis after 8 days can be explained as a consequence of cell division in the prevascular tissue. I n this w a y histophotometric and biochemical D N A determinations are in good agreement. Our results show t h a t light not only reduces the formation of polyploid nuclei, it also has a considerable effect on the nuclear volume. A linear correlation between the logarithm of the a m o u n t of D N A and the logarithm of the nuclear volume is well k n o w n in plants as well as in animals (Alvarez, 1969; Kastcn, 1965; Schreiber et al., 1969). The nuclear volume gives an indication for cell activity (StSckert, 1962). Obviously the epicotyl nuclei in the light are physiologically as inactive as the plumula nuclei in dark-grown seedlings. This is in agreement with biochemical determinations of the ratio R N A / D N A . This ratio increases strongly in the dark but remains practically constant in the light. F u r t h e r m o r e in the dark this ratio reaches a m a x i m u m coinciding with a maximal nuclear swelling after 3 or 4 days of germination. These results support the hypothesis t h a t cell elongation growth and R N A synthesis are enhanced b y endopolyploidy (Nagl, 1973; Scharpe and Van Parijs, 1973). Endomitosis can not be considered as an obligatory process in cell elongation, but cells becoming polyploid can enhance the elongation capacity of the organ. I n agreement with this hypothesis a hormone such as gibbcrellin can enhance cell elongation within a fixed range, determined by the polyploidy level of the tissue. This conclusion seems to be confirmed by another series of experiments, in which endomitosis was blocked b y very short red-light irradiations (660 nm) at the same level of polyploidy as in the light, without blocking cell elongation to the same degree as in the light (Boeken et al., 1975).
References Alvarez, M. g . : Cytophotometric study of nuclear proteins and nucleic acids in parenchyma-
tous tissue of the orchid embryo. Exp. Cell Res. 57, 179-184 (1969) Boeken, G., Van Oostveldt, P., Van Parijs, 1~., Fredericq, H.: Phytochrome-controlled endomitosis during the process of cell elongation in the epicotyl of Pisum sativum seedlings. Arch. internat. Physiol. Biochem. 83, in press (1975) Bopp, lV[.: ttemmung des Streckungswachstums etiolierter Sprossachsen durch FUDI~. Z. Pflanzenphysiol. 57, 173-187 (19673) Bopp, M. : Internodienstreckung bei dikotylen Pflanzcn und D:NS-Synthese. Exp. Cell l~es. 48, 218-221 (1967b) Bopp, M. : Ubcr den Einbau von aH-Thymidine in etiolierenden Sprossachsen. Z. Pflanzenphysiol. 63, 10-14 (1970) Burmaster, C.F.: Microdetermination of u- and fl-glycerophosphates. J. biol. Chem. 164, 233-240 (1946) Burton, K. : A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of DNA. Biochem. J. 62, 315-323 (1955) Capesius, I., Bopp, M. : Ober die Synthese yon DNS in den Keimpflanzen yon Sinapis alba L. Planta (Berl.) 94, 220-228 (1970)
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Capesius, I., Bopp, M., Claus, W. : Die lag-Phase bei der FUDR-Wirkung auf DNA-Synthese und Streckungswachstum bei Sinapis alba. Planta (Berl.) 103, 65-73 (1972) Capesius, I., StShr, M.: Endopolyploidisierung wi~hrend des Streckungswachstums der ttypokotyle von Sinapis alba. Protoplama 82, 147-153 (1974) Cassie, R. M. : Some uses of probability paper in the analysis of size frequency distributions. Austral. J. Mar. Fresh Water Res. 5, 513-522 (1954) Crow, E. L., Davis, F. A., Maxfield, M.W.: Statistics Manual. NewYork: Dover Publ. (1960) Degani, Y., Atsmon, D. : Enhancement of non nuclear DNA synthesis associated with hormone induced elongation in the cucumber hypocotyl. Exp. Cell Res. 61, 226-229 (1970) De Greef, J., Frederieq, H.: Photomorphogenic and chlorophyll studies in the bryophyte Marchantia polymorpha. II. Photobiological responses to terminal irradiations with different red/far-red ratios. Physiol. Plantarum 22, 462468 (1969) Giles, K. W., Myers, A. : The role of nucleic acids in the growth of the hypocotyl of Lupinus albus under varying light and dark regimes. Biochem. biophys. Aeta (Amst.) 87, 460477 (1964) Kasten, F. H. : The potential of quantitative cytochemistry in tumor research. In: Introduction to quantitative cytochemistry, vol. 2, p. 263-296, Wied, G.L., Bahr, G.F., eds. New York-London: Acad. Press 1970 Lison, L.: tIistochemie et eytochemie animales. Paris: Gauthier-Villars 1953 Logan, J. E., Marmell, W. A., Rossiter, R. J. : A note on the determination of DNA and I~NA in tissue from the nervous system by UV absorption. Biochem. J. 51, 480482 (1952) Low, V. H. K. : Effects of light and darkness on the growth of peas. Austral. J. biol. Sci. 24, 187-195 (1971) MSrike, K. D. : Mathematische ErSrterungen zur Megmethodik yon nieht runden Zellkernen. Anat. Anz. 100, 87-99 (1953) Nagl, W. : The Mitotic and endomitotic nuclear cycle in Allium carcinatum. IV. ~H-Uridine incorporation. Chromosoma (Berl.) 44, 203-212 (1973) Nitsan, J., Lung, A.: Inhibition of cell division and cell elongation in higher plants by inhibitors of DNA replication. Develop. Biol. 12, 358-376 (1965) Ogur, M., I~osen, G. : The nucleic acids of plant tissues. Arch. Biochem. 24, 262-276 (1950) Busch, E. : DNA cytophotometry of salivary fland nuclei and other tissue systems in dipteran larvae. In: Introduction to quantitative cytoehemistry, vol. 2, p. 357-397, Wied, G.L., Bahr, G. F., eds., New York-London: Acad. Press 1970 Sandritter, W.: Ultraviolettmikrospektrophotometrie. In: Handbuch der Histochemie, vol. 1, pt. 1, p. 220-338, Graumann, W, Neumann, K., eds., Stuttgart: Fischer 1958 Scharpe, A., Van Parijs, B. : The formation of polypoid cells in ripening cotyledones of Pisum sativum L. in relation to ribosome and protein synthesis. J. exp. Bog. 24, 216-222 (1973) Schneider, W. C.: Phosphorus compounds in animal tissues. I. Extraction and estimation of deoxypentose nucleic acid and of pentose nucleic acid. J. biol. Chem. 161,293-303 (1945) Schreiber, G., Amorin, F. M. 0., Cavenaghi, T. M., Fallieri, L.A., Gerken, S.E., MeLueci, N., Sang'Anna, Y.X., Sehreiber, M. t~.: Significance of the ratio "DNA/nuclear size" in the differentiation of tissues. Genetics 61, Suppl. 9, 161-170 (1969) St/Scker, E. : Autoradiographische Untersuchungen zur l~ibonukleins~ure-tmd Eiweigsynthese im nuklearen Funktionsformwechsel der exokrinen PaIfl~reas-Zelle. Z. Zellforsch. 57, 145-171 (1962) Stockx, J., Vandendriessche, L.: Synthesis and properties of 1-1'-diglyceromonophosphoric acid. Bull. Soc. Chem. Belg. 65, 919 927 (1956) Van Parijs, 1%.: Quantitative changes of ribonueleic acid (RNA), desoxyribonucleic acid (DNA), protein and dry matter in different organs of pea seedlings, during germination and cell elongation. Arch. internat. Physiol. Biochem. 75, 125-138 (1967) Van Parijs, t~., Vandendriessche, L. : Changes of the DNA content of nuclei during the process of cell elongation in plants. I. The formation of polytene chromosmes. Arch. int. Physiol. Biochim. 74, 579-586 (1966a) Van Parijs, P~., Vandendriessche, L. : Changes of the DNA content of nuclei during the process of cell elongation in plants. II. Variations other than doublings of the amount of Feulgen-stain in maturing plant cells. Arch. int. Physiol. Bioehim. 74, 587-591 (1966b)
Effect of Light on Nucleic-acid Synthesis and Polyploidy Level in Elongating Epicotyl Cells of Pisum sativum P. Van Oostveldt and R. Van Parijs Laboratory of Biochemistry, Faculty of Agricultural Sciences, State University, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium Received 27 January; accepted 25 March 1975
Summary. The synthesis of DNA and RINA and the increase in dry matter were followed in the elongating cells of the epicotyl of peas (Pisum sativum L.) germinating in total darkness or in continuous light. The amounts of DNA and I%NA per epicotyl were estimated by chemical methods, the amount of DNA per cell was measured by histophotomctric techniques. The increase in DI~A, t%I~TAand dry matter in the epicotyl cells is much higher during germination in darkness than in light. During elongation in the dark most cortical epicotyl cells reach the 8C polyploidy level, in the light only the 4C polyploidy level is reached. A decrease in RNA synthesis is in agreement with a reduction in nuclear volume.
Introduction The metabolism of nucleic acids (NAs) during cell elongation is a widely studied problem of plant physiology. A causal relationship between DNA synthesis and cell elongation was studied especially in two ways. In one type of experiments, tissues were treated with a plant hormone, such as gibberellin, which promoted cell elongation, and the enhanced DNA synthesis was followed by [all] thymidine incorporation (e.g. Degani and Atsmon, 1970). Other workers used light as an external factor which inhibited cell elongation ; the reduced DNA synthesis was again followed by [aH]thymidine incorporation (e.g. Bopp, 1970). In either type of experiment the causal relationship between DI~A synthesis and cell elongation was studied by blocking DNA synthesis with 5-fluorodeoxyuridine and observing the inhibitory effect on cell elongation (Nitsan and Lang, 1965; Bopp, 1967a; Capesius et al., 1972). I n most cases cpicotyls or hypocotyls of germinating seeds were used. Most authors assumed t h a t the growth of epicotyl or hypocotyl occurs only by cell elongation without cell divisions. This implies t h a t DNA synthesis is restricted to extranuclcar DNA synthesis or to endomitotic DNA duplication. However, direct histophotometric estimations of the amount of DNA per cell, in order to determine the level of endomitosis, were rarely done (Capesius and StShr, 1974). Van Parijs (1967) followed the total amount of DNA, RNA, protein and dry matter during elongation in darkness of the epicotyl of Pisum sativum and established an endomitotic cell cycle (Van Parijs and Vandendriessche, 1966a). In this paper we report chemical determinations of Rb~A and DNA amounts in epicotyls of pea seedlings growing either in darkness or in light, together with histophotometric DNA measurements and deter-
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m i n a t i o n s of t h e n u c l e a r v o l u m e . I t w a s o u r i n t e n t i o n t o see w h e t h e r t h e i n h i b i t o r y e f f e c t of l i g h t o n cell e l o n g a t i o n is a c c o m p a n i e d b y a n i n h i b i t i o n of t h e t o t a l a m o u n t of N A s a n d b y a n i n h i b i t i o n i n t h e e n d o p o l y p l o i d y l e v e l of t h e e p i c o t y l ceils. Material and Methods
Plant Material. Seeds of the common garden pea (Pisum sativum L., cv. Rondo;SeedTrade Labor, Ghent, Belgium) were soaked in running tap water for 18 h. The epicotyls (internode between cotyledons and first leaf) were excised directly after soaking (0-day-old seedling) and after different periods of further germination. The seeds were germinated a t 15 ~ in plastic dishes filled with moistened polyester foam. A t each sampling, a b o u t 200 epicotyls were excised, collected in glass vials with dry ice, and stored in a freezer a t -- 15 ~ until use. For germination in continuous light the dishes were placed under a set of 10 fluorescent lamps ( " P h y t e r " ; ACEC, Charleroi, Belgium). The spectral energy distribution of these lamps is published in De Greef and Fredericq (1969). Light intensity at the seed level is 19 ~zW cm -2 n m at 650 n m a n d 7 ~W cm -2 n m at 450 nm. Extraction o/Nucleic Acids. The excised epicotyls were ground in a mortar with dry ice and further homogenized in a Teflon-glass Potter-E1vehjem homogenizer at 0 ~ After centrifugation (5000 • g for 10 min) the pellet was washed several times according to Ogur and l~osen (1950) to remove non-NA material. The NAs were extracted from the washed residue by heating in 5% trichloroacetic acid at 90 ~ for 15 min (Schneider, 1945). The total NA content of the extract was determined by U V absorption a t 268.5 n m (Logan et al., 1952) and b y phosphate determination after destruction of the NAs in 10 N HeSO 4 following the method of Burmaster (1946) as adapted by Steckx and Vandendriessche (1956). DNA was measured according to B u r t o n (1956) using deoxyribose in 5 % TCA as standard. R N A was calculated from the difference between the total ~ A content and the DNA content. The amounts of DNA and R N A were expressed as DNA-phosphorus (DNA-P) and RNAphosphorus (RNA-P). All measurements were done in quadruple. Dry Matter. A p a r t of the homogenate in 95 % ethanol was first dried a t low temperature, then at 100 ~ for 18 h. Histophotometric DNA Determination. Whole embryos, epicotyls or parts of the epicotyl were excised, fixed in A F A (ethanol 95?/o-formaldehyde 37%-glacial acetic acid, 75:20: 5, v/v) for 18 h at room temperature, dehydrated through tertiary butanol, and embedded in paraffin. Sections were cut a t 15 ~m for 0-day-old seedlings and at 25 [zm for 4- and 8day-old seedlings. Tissues of different stages were fixed, hydrolysed, and stained simultaneously in order to obtain comparable relative amounts of DNA per cell. Feulgen staining was applied after hydrolysis with 1N HC1 for 12 rain at 60 ~ The DNA content of the cortex nuclei was determined by the cytophotometric technique of Lison (1953, p. 139), using a multiple-plug measurement technique. I n this method a diaphragm is first directed beside the nucleus and a blank is read (transmission 100% ). Subsequently, different transmission readings (T) are t a k e n in the nucleus. The a m o u n t of DNA is expressed as log 1/T • area of the nucleus. The area of the nucleus is estimated b y planimetry. The transmissions were measured a t 556 n m (interference filter Filtraflex B-40; Balzer, Liechtenstein) in the nuclei of the epicotyl cortex, after 0, 4 and 8 days of germination. The plumule of the 4-day-old seedling (germination in the dark) was included as reference (diploid a m o u n t of DNA). Statistical Methods. Volumes were calculated from the formula of Puff (M6rike, 1953), i.e. V = 0.8488 F~/L wherein F ~ the area of the projected image of the nucleus, L = the length of the long axis. Regression lines were calculated on a programmed calculator (Diehl, Nfirnberg, Germany). Multimodal distributions of the a m o u n t of D•A per nucleus were separated in different unimodal populations by plotting the cumulative frequency diagram on probability paper according to the method of Cassie (1954). The percentages of the nuclei in the different populations were estimated b y considering the points of inflection of the curves (Rasch, 1970).
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Results
Chemical Determination o] Nucleic Acids and Dry Matter Nucleic-acid determinations were carried out from the 3rd day on. As shown by Van Parijs (1967) no synthesis of NA is occurring during the first 2 days. Starting with a net value of 0.23 ~g DNA-P per epicotyl a value of 0.82 and 0.62 9g DNA-P per epicotyl is reached in continuous dark and in continuous light, respectively (Fig. i). As can be seen in the figure this difference is highly significant. Thus light inhibits DNA synthesis. If the growth of the epicotyl is occurring by cell enlargement only, then we can assume that light reduces endomitosis. The amount of total NA-phosphorus per epicotyl reaches a m a x i m u m after 3 days, in the dark as well as in the light (Fig. 2). No significant differences are found between the total NA content as estimated from UV absorption (Logan et al., 1952) and from phosphorus determinations (Burmastcr, 1946). The ratio of RNA/DNA gives an indication for the amount of I~NA per cell (Fig. 3). This ratio reaches a maximum after 3 days of germination in the dark, but no clear m a x i m u m is found in the light. This difference between light- and dark-germinated seedlings is significant at the 5% level as was deduced from a non-parametric test. This test was used because the estimation of the standard deviation from a series of ratios is not appropriate. The test can be formulated as t--
Wl @ W2
wherein 21 and 2~ are the means of the two groups of ratios and wa and w~ are the ranges respectively of group 1 and group 2. The value t is compared with the value found in table (Crow et al., 1960). The I~NA/DNA ratio in the dark, after full elongation at 8 days, is nearly the same as the ratio I%NA/DNA in the light. The amount of dry matter of the epicotyl continues to increase at the time the amount of RNA per cell reaches a minimum. Finally the amount of dry matter in the light-grown epicotyl is about half the amount in the dark-grown epicotyl.
Histophotometric D N A Determinations in the Cortex o/ the Epicotyl 1. Polyploidy. The amounts of DNA, as measured by the Feulgen procedure, are shown in Table 1 and Fig. 4. As was to be expected from the results obtained with chemical methods the histophotometric determinations show t h a t most nuclei reach the oetaploid 8 C DNA level after 4 days of germination in the dark. After the same period of germination under continuous light, most of the nuclei have reached only the tetraploid DNA level. After 8 days of germination either in the dark or under continuous light, the DNA level per nucleus seems to be stabilized and there are no obvious differences between the situation after 4 days or after 8 days of germination. 2. Relation between D N A per Nucleus and Nuclear Volume. When the logarithm of the relative amount of DNA per nucleus is plotted against the logarithm of the nuclear volmne, a clear linear correlation is observed. From such plots (diagraphs) we calculated the relative nuclear volume corresponding to each
290
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Nucleic-acid Synthesis and Light
291
Table 1. Percentages of nuclei of different ploidy levels in cortex cells at different stages of epicotyl elongation Days, light conditions
2C
4C
8C
16C
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23 81 9O 14 19
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292
P. Van Oostveldt and R. Van Parijs
Table 2. Correlation analysis between the logarithm of the nuclear volume (y) and the logarithm of the amount of DNA (x) Relative nuclear volumes are calculated for 2C, 4C and 8C nuclei of the cortex of epieotyls germinating in the dark or in the light. Plumule nuclei are ehoosen as a reference Tissue, light conditionsa
Relative nuclear volume 2C
4C
8C
P1 4D Ep 4D Ep 8D Ep 0D Ep 4L Ep 8L
1 4.5 2.8 1.2 0.71 1.3
1.8 7.3 4.7 1.9 1.6 2.0
3.5 11.2 7.7 3.0 3.6 2.8
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0.90706 0.61937 0.74112 0.64194 1.11491 0.50344
0.10298 0.13170 0.07571 0.08288 1.14342 0.19007
0.66473 0.42908 0.62434 0.69560 0.66798 0.25847
100 100 152 66 77 100
8.81 4.70 9.79 7.74 7.77 2.65
a P1 = plumule, Ep = epicotyl ; 0 = 0 days, 4 = 4 days, 8 = 8 days; D = continuous darkness, L = continuous light. b by=+x = regression coefficient of y from x. e ay+x = standard deviation of by_+x . d r = correlation coefficient. e N = total amount of nuclei measured. f t = Student's t for the correlation coefficient.
polyploidy level in order to get some i n f o r m a t i o n a b o u t the a c t i v i t y of the nuclei. The results of these calculations are shown in Table 2. Since the diploid nuclei from 4-day-old dark-grown p l u m u l e s have the smallest volume, t h e y can be considered as physiologically less active (St6ekert, 1962). These nuclei were used as reference (volume = 1). I n the dark-grown tissues the nuclei, as deduced from their relative volume, seem to be more active t h a n in the light-grown tissue. 3. Changes, Other than D o u b l i n g s , i n the A m o u n t o / D N A per N u c l e u s . Earlier histophotometrie D N A d e t e r m i n a t i o n s have shown t h a t the diploid cortex cells of the epicoty] before g e r m i n a t i o n c o n t a i n a b o u t 20 % less D N A t h a n the diploid meristematie cells of the p l u m u l e (Van Parijs a n d Vandendriessehe, 1966b) a n d the same difference was f o u n d between 8 C nuclei of young, n o t fully elongated cortex cells a n d 8 C nuclei of fully elongated cortex cells. This m e a n s t h a t in the y o u n g 8C nuclei of cells which prepare for elongation, some D N A is missing b u t is present later when the cells are fully elongated. I n our results (Fig. 4) a n analogous difference is found at the 4C D N A level between the cortex cells of 4- a n d of 8-day-old epieotyls grown i n the light. The fact t h a t this difference is f o u n d during g e r m i n a t i o n in the light as well as i n the dark indicates t h a t influences of physical errors, such as d i s t r i b u t i o n a l error or the SehwartzschildVilliger effect (Sandritter, 1958) which are d e p e n d e n t on the e x t i n c t i o n values, are n o t very likely. The e x t i n c t i o n of the F e u l g e n stain (DNA per v o l u m e ; Table 2) in nuclei of light- a n d dark-grown cortex varies b y a factor 2 or 3. B u t the differences in the a m o u n t of F e n l g e n stain between nuclei at the b e g i n n i n g a n d those at the end of elongation are f o u n d in dark as well as in light. More evidence for the absence of some D N A fraction a n d indications a b o u t the n a t u r e of this D N A will be presented in a n o t h e r publication.
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Discussion Our results show t h a t inhibition of cell elongation by light in the epicotyl of Pisum sativum is accompanied by a reduction of endomitosis in the cortex cells. This process of endomitosis is completely finished after 4 days of germination and precedes growth in length of the epicotyl since elongation is closely parallel to dry-matter increase (van Parijs, 1967). The inhibition of cell elongation by light involves also a reduced synthesis of I~NA. In contrast to the behaviour of Lupinus albus hypoeotyls (Giles and Myers, 1964) we noted a sharp decrease in the total amount of RNA per epicotyl in Pisum sativum after 4 days in the dark. I t m a y be argued that the difference in the metabolism of RNA (especially synthesis of ribosomes) between Lupinus and Pisum is related to differences in the germination physiology. In Pisum the elongation of the epieotyl m a y be followed by the elongation of the higher internodes up to the sixth (Low, 1971). RNA synthesized in the epicotyl is supposed to break down and to be transported into the higher internodes. In Lupinus, the hypocotyl is the only internode which is growing in the dark; thus in this case RNA is needed during the whole germination process. This hypothesis is supported by the fact that transfer of Lupinus seedlings from dark to light causes a drastic decrease in the amount of RNA (Giles and Myers, 1964). The hydrolyzed RNA can then be used in the higher internodes which are elongating in the light. A number of investigators demonstrated a relationship between DNA synthesis and cell elongation (Nitsan and Lang, 1965; Bopp, 1967a, 1970; Capesius et al., 1972). However in most eases these studies did not include histophotometrie DNA determinations and therefore it was not possible to conclude if DNA synthesis was restricted to endomitosis. Only recently Capesius and St6hr (1974) have shown t h a t inhibition of cell elongation by 5-fluorodeoxyuridine is accompanied by a reduced endomitotie cell cycle. In germinating seeds the process of endomitotie DNA synthesis seems to occur very early. In Sinapis alba most DNA replication is finished 60 h after germination (Capesius and Bopp, 1970; Capesius and StShr, 1974). Light as an external factor can irreversibly block this DNA synthesis (Capesius and Bopp, 1970). I n our experiments DNA synthesis was slowed down by growing the seedlings at 15 ~ Nevertheless after 4 days, at the stage the epieotyls have reached half their final length, the endomitotic activity was finished. In older material (8-10 days) a number of cells are dividing mainly in the prevaseular tissue where adventious roots arise. Van Parijs and Vandendriessche (1967 a) carried out DNA measurements in the prevascular tissue and found t h a t these nuclei were blocked at a 4C DNA level after 4 to 8 days, probably corresponding to nuclei in a premitotic G 2 phase. From transverse sections of epicotyls (8-day-old) we found that about 2500 cortex cells and 1500 cells of the prevascular elements are present in the epicotyl. Epidermis cells were not counted. If we know the amount of DNA at 0 days, then we can calculate the total amount of DNA after 4 days germination in light or dark, assuming that all the 1500 prevascular cells become tetraploid and that the cortex cells reach the levels of polyploidy shown in Table 1. In this way we find t h a t in the light total amount of DNA
294
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will be 0.45 ~g. Experimentally, a value of 0.65~zg is found. For germination in the dark we calculate an a m o u n t of D N A of 0.68 ~g while experimentally a value of 0.80 ~zg is found. The calculated ratio of the a m o u n t D N A per epicotyl in the dark and the a m o u n t of D N A per epicotyl in the light, equals 1.50 while experimentally a ratio of 1.23 is found. A Z 2 test shows t h a t the calculated amounts of DNA are in agreement with the experimental results. The enhanced D N A and RIgA synthesis after 8 days can be explained as a consequence of cell division in the prevascular tissue. I n this w a y histophotometric and biochemical D N A determinations are in good agreement. Our results show t h a t light not only reduces the formation of polyploid nuclei, it also has a considerable effect on the nuclear volume. A linear correlation between the logarithm of the a m o u n t of D N A and the logarithm of the nuclear volume is well k n o w n in plants as well as in animals (Alvarez, 1969; Kastcn, 1965; Schreiber et al., 1969). The nuclear volume gives an indication for cell activity (StSckert, 1962). Obviously the epicotyl nuclei in the light are physiologically as inactive as the plumula nuclei in dark-grown seedlings. This is in agreement with biochemical determinations of the ratio R N A / D N A . This ratio increases strongly in the dark but remains practically constant in the light. F u r t h e r m o r e in the dark this ratio reaches a m a x i m u m coinciding with a maximal nuclear swelling after 3 or 4 days of germination. These results support the hypothesis t h a t cell elongation growth and R N A synthesis are enhanced b y endopolyploidy (Nagl, 1973; Scharpe and Van Parijs, 1973). Endomitosis can not be considered as an obligatory process in cell elongation, but cells becoming polyploid can enhance the elongation capacity of the organ. I n agreement with this hypothesis a hormone such as gibbcrellin can enhance cell elongation within a fixed range, determined by the polyploidy level of the tissue. This conclusion seems to be confirmed by another series of experiments, in which endomitosis was blocked b y very short red-light irradiations (660 nm) at the same level of polyploidy as in the light, without blocking cell elongation to the same degree as in the light (Boeken et al., 1975).
References Alvarez, M. g . : Cytophotometric study of nuclear proteins and nucleic acids in parenchyma-
tous tissue of the orchid embryo. Exp. Cell Res. 57, 179-184 (1969) Boeken, G., Van Oostveldt, P., Van Parijs, 1~., Fredericq, H.: Phytochrome-controlled endomitosis during the process of cell elongation in the epicotyl of Pisum sativum seedlings. Arch. internat. Physiol. Biochem. 83, in press (1975) Bopp, lV[.: ttemmung des Streckungswachstums etiolierter Sprossachsen durch FUDI~. Z. Pflanzenphysiol. 57, 173-187 (19673) Bopp, M. : Internodienstreckung bei dikotylen Pflanzcn und D:NS-Synthese. Exp. Cell l~es. 48, 218-221 (1967b) Bopp, M. : Ubcr den Einbau von aH-Thymidine in etiolierenden Sprossachsen. Z. Pflanzenphysiol. 63, 10-14 (1970) Burmaster, C.F.: Microdetermination of u- and fl-glycerophosphates. J. biol. Chem. 164, 233-240 (1946) Burton, K. : A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of DNA. Biochem. J. 62, 315-323 (1955) Capesius, I., Bopp, M. : Ober die Synthese yon DNS in den Keimpflanzen yon Sinapis alba L. Planta (Berl.) 94, 220-228 (1970)
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Capesius, I., Bopp, M., Claus, W. : Die lag-Phase bei der FUDR-Wirkung auf DNA-Synthese und Streckungswachstum bei Sinapis alba. Planta (Berl.) 103, 65-73 (1972) Capesius, I., StShr, M.: Endopolyploidisierung wi~hrend des Streckungswachstums der ttypokotyle von Sinapis alba. Protoplama 82, 147-153 (1974) Cassie, R. M. : Some uses of probability paper in the analysis of size frequency distributions. Austral. J. Mar. Fresh Water Res. 5, 513-522 (1954) Crow, E. L., Davis, F. A., Maxfield, M.W.: Statistics Manual. NewYork: Dover Publ. (1960) Degani, Y., Atsmon, D. : Enhancement of non nuclear DNA synthesis associated with hormone induced elongation in the cucumber hypocotyl. Exp. Cell Res. 61, 226-229 (1970) De Greef, J., Frederieq, H.: Photomorphogenic and chlorophyll studies in the bryophyte Marchantia polymorpha. II. Photobiological responses to terminal irradiations with different red/far-red ratios. Physiol. Plantarum 22, 462468 (1969) Giles, K. W., Myers, A. : The role of nucleic acids in the growth of the hypocotyl of Lupinus albus under varying light and dark regimes. Biochem. biophys. Aeta (Amst.) 87, 460477 (1964) Kasten, F. H. : The potential of quantitative cytochemistry in tumor research. In: Introduction to quantitative cytochemistry, vol. 2, p. 263-296, Wied, G.L., Bahr, G.F., eds. New York-London: Acad. Press 1970 Lison, L.: tIistochemie et eytochemie animales. Paris: Gauthier-Villars 1953 Logan, J. E., Marmell, W. A., Rossiter, R. J. : A note on the determination of DNA and I~NA in tissue from the nervous system by UV absorption. Biochem. J. 51, 480482 (1952) Low, V. H. K. : Effects of light and darkness on the growth of peas. Austral. J. biol. Sci. 24, 187-195 (1971) MSrike, K. D. : Mathematische ErSrterungen zur Megmethodik yon nieht runden Zellkernen. Anat. Anz. 100, 87-99 (1953) Nagl, W. : The Mitotic and endomitotic nuclear cycle in Allium carcinatum. IV. ~H-Uridine incorporation. Chromosoma (Berl.) 44, 203-212 (1973) Nitsan, J., Lung, A.: Inhibition of cell division and cell elongation in higher plants by inhibitors of DNA replication. Develop. Biol. 12, 358-376 (1965) Ogur, M., I~osen, G. : The nucleic acids of plant tissues. Arch. Biochem. 24, 262-276 (1950) Busch, E. : DNA cytophotometry of salivary fland nuclei and other tissue systems in dipteran larvae. In: Introduction to quantitative cytoehemistry, vol. 2, p. 357-397, Wied, G.L., Bahr, G. F., eds., New York-London: Acad. Press 1970 Sandritter, W.: Ultraviolettmikrospektrophotometrie. In: Handbuch der Histochemie, vol. 1, pt. 1, p. 220-338, Graumann, W, Neumann, K., eds., Stuttgart: Fischer 1958 Scharpe, A., Van Parijs, B. : The formation of polypoid cells in ripening cotyledones of Pisum sativum L. in relation to ribosome and protein synthesis. J. exp. Bog. 24, 216-222 (1973) Schneider, W. C.: Phosphorus compounds in animal tissues. I. Extraction and estimation of deoxypentose nucleic acid and of pentose nucleic acid. J. biol. Chem. 161,293-303 (1945) Schreiber, G., Amorin, F. M. 0., Cavenaghi, T. M., Fallieri, L.A., Gerken, S.E., MeLueci, N., Sang'Anna, Y.X., Sehreiber, M. t~.: Significance of the ratio "DNA/nuclear size" in the differentiation of tissues. Genetics 61, Suppl. 9, 161-170 (1969) St/Scker, E. : Autoradiographische Untersuchungen zur l~ibonukleins~ure-tmd Eiweigsynthese im nuklearen Funktionsformwechsel der exokrinen PaIfl~reas-Zelle. Z. Zellforsch. 57, 145-171 (1962) Stockx, J., Vandendriessche, L.: Synthesis and properties of 1-1'-diglyceromonophosphoric acid. Bull. Soc. Chem. Belg. 65, 919 927 (1956) Van Parijs, 1%.: Quantitative changes of ribonueleic acid (RNA), desoxyribonucleic acid (DNA), protein and dry matter in different organs of pea seedlings, during germination and cell elongation. Arch. internat. Physiol. Biochem. 75, 125-138 (1967) Van Parijs, t~., Vandendriessche, L. : Changes of the DNA content of nuclei during the process of cell elongation in plants. I. The formation of polytene chromosmes. Arch. int. Physiol. Biochim. 74, 579-586 (1966a) Van Parijs, P~., Vandendriessche, L. : Changes of the DNA content of nuclei during the process of cell elongation in plants. II. Variations other than doublings of the amount of Feulgen-stain in maturing plant cells. Arch. int. Physiol. Bioehim. 74, 587-591 (1966b)
