Ribosome Synthesis in Greening Primary Leaves of Bean Seedlings (Phaseolus vulgaris)

Biochem. Physiol. Pflanzen (BPP), Bd. 162, S. 245-264 (1971) Institute of Genetics, University of Copenhagen

Ribosome Synthesis in Greening Primary Leaves of Bean Seedlings (Phaseolus vulgaris) By KIRSTEN S0GAARD ANDERSEN With 6 figures and plate 13-17 (Received November 11, 1970)

Summary Sixteen day old dark grown bean seedlings, excised below the cotyledons, were fed tritiated uridine during 45 hours of illumination. Quantitative electron microscopic radioautography of the paraenchyma cells from primary leaves, revealed that 45 per cent of the label was located in chloroplasts, 45 per cent in the cytoplasm and 8 per cent in nuclei, with the highest amount of label per unit area in nucleoli. In 11 day old dark grown leaves doubling of the DNA and RNA content was registered after 45 hours of light. When 11 day old dark grown seedlings, at different stages of chloroplast development, were supplied with sucrose and ammonium citrate, plastid development and nucleic acid accumulation proceeded up to five hours unimpaired by the excision. Such seedlings were supplied with carbon-14-labeled uridine, and ribosomes were isolated from the primary leaves and studied by sucrose gradient centrifugation. Both cytoplasmic and chloroplastic ribosomes could be labeled throughout the period of chloroplast development. The degree of ribosomal labeling was slightly higher in the chloroplasts than in the cytoplasm during the period of grana formation.

1. Introduction

Chloroplastic and cytoplasmic ribosomes can be distinguished. LYTTLETON (1962) observed that spinach chloroplasts contained ribosomes with a lower sedimentation coefficient (70S) than cytoplasmic ribosomes (80S). The same result was obtained with photosynthetic organisms ranging from algae to higher plants (Euglena EISENSTADT and BRAWERMAN 1964, Chlamydomonas HOOBER and BLOBEL 1969, Brassica CLARK, MATTHEWS and RALPH 1964, Pisum SISSAKIAN, FILIPPOVICH, SVETAILO and ALIYEV 1965, Nicotiana BOARDMAN, FRANCKI and WILDMAN 1966, Phaseolus STUTZ and NOLL 1967). Chloroplastic ribosomes of higher plants contain 23S and 16S RNA as compared to the 25S and 18S RNA of the cytoplasmic ribosomes (LOENING and INGLE 1967; STUTZ and NOLL 1967). Failure to detect the 23S RNA of chloroplastic ribosomes e. g. in spinach (SPENCER and WHITFELD 1966) can be due to degradation of the RNA during its isolation (INGLE 1968a).

246

K. S.

ANDERSEN

To determine the site of chloroplastic ribosomal-RNA synthesis, hybridization has been tried between ribosomal-RNA species and DNA species isolated from cell organelle fractions. The DNA species were separated by cesium chloride density gradient centrifugation (Euglena SCOTT and SMILLIE 1967, Nicotiana TEWARI and WILDMAN 1968). Chloroplastic ribosomal-RNA appeared to hybridize specifically with both chloroplastic and nuclear DNA, whereas cytoplasmic ribosomal-RNA hybridized specifically only with nuclear DNA. Difficulties in determining unequivocally the origin of the DNA species used for the hybridization experiments (WELLS and BIRNSTIEL 1969) as well as specificity problems of the hybridization technique (INGLE, WELLS and POSSINGHAM 1969) do not yet permit a definite answer whether both. chloroplastic and nuclear DNA code for chloroplastic ribosomal-RNA. GIBBS (1969) analysed by radioautography the labeling pattern of cell organelles in the alga Ochromonas danica, which was supplied with tritiated uridine at the time of chloroplast development. The RN-ase removable label appeared first in nucleoli and in DNA-containing regions of chloroplasts. Cytoplasm and chloroplast stroma became labeled only after a prolonged period. These results are consistent with cytoplasmic ribosomal-RNA synthesis at the nucleolus organizer and chloroplastic ribosomal-RNA synthesis at the chloroplastic DNA. Little information is available on synthesis of chloroplastic ribosomes and chloroplastic ribosomal-RNA in relation to chloroplast development (review cf. SMILLIE and SCOTT 1969). In Euglena (BRAWERMAN, POGO and CHARGAFF 1962; BRAWERMAN 1963) measurement of RNA isolated from chloroplast fractions and from whole cells using dark grown and greened cultures reveals synthesis of both cytoplasmic and chloroplastic RNA during chloroplast development, the major part of the RNA being ribosomal. ZELDIN and SCHIFF (1967, 1968) followed the synthesis of cytoplasmic and chloroplastic RNA in Euglena by 32P-labeling. In cotyledons of radish (Raphanus sativus) INGLE (1968a and b) has shown that during the first four days after sowing the cotyledons accumulate chloroplastic ribosomalRNA in the light at three times the rate of the accumulation found in the dark, the rates in light and dark of cytoplasmic ribosomal-RNA accumulation being equal. INGLE incubated the light grown cotyledons in 32P-orthophosphate and found a maximum labeling of chloroplastic ribosomal-RNA on the third day after sowing. In Phaseolus vulgaris GYLDENHOLM (1968) observed a 50 per cent increase in chloroplast-associated RNA during 45 hours greening of dark grown primary leaves, the increase being approximately half the total increase of RNA. BOARDMAN (1966) found 0.14 mg 70S ribosomes per primary leaf in dark grown bean plants vs. 0.23 mg in green house grown plants, and 0.38 mg 80S ribosomes per leaf in dark grown vs. 0.50mg in green house grown plants. In the present paper an attempt has been made to determine the cellular distribution of the RNA synthesized during greening of dark grown primary bean leaves.

Ribosome Synthesis in Greening Primary Leaves of Bean Seedlings etc.

247

The dark grown leaves were fed tritiated uri dine during greening. Quantitative radioautographic analysis revealed the labeling pattern of the cell organelles whereas the distribution of label among the 16-25S ribosomal- and the 4-5S soluble-RNA was determined by isolation of native RNA. Furthermore it was attempted to follow the synthesis of chloroplastic and cytoplasmic ribosomes in relation to plastid development. This was done by feeding labeled uri dine to dark grown bean plants at different times after the light induced onset of plastid development, followed by isolation and analysis of the labeled ribosomes from the primary leaves of the plants. 2. Materials and methods

a) Plant material Seeds of Phaseolus vulgaris var. Alabaster (Weibull, Sweden) were sterilized for 30 minutes in a 1 per cent sodium hypochlorite solution, rinsed in tap water, and sown in moistened vermiculite. Plants were grown in the dark for 11 or 16 days at 25 DC. Chloroplast development was then initiated at 24-25 DC by illumination (ca. 3000 lux) of the dark grown leaves with 2 Philips TL 20W/33 and 1 Sylvania GRO LUX 20 W tubes at a distance of 15 cm from leaves (cf. GYLDENHOLM 1968).

b) RNA labeling Distal segments of plants were excised three centimeters below the cotyledons and placed with their stems in small tubes containing 0.1 ml medium. The basal one cm portion of the stems were then cut off under solution and removed, and the remaining segments with cotyledons and primary leaves (hereafter called plant segments) were kept at a relative humidity of approximately 95 per cent. The medium consisted of 0.1 mM uridine (La Roche & Co.), 0.1 M sucrose, 0.1 mM ammonium citrate in tap water. For the RNA labeling experiments the uridine was replaced by uridine- 14 C-(U) (Amersham, England). When most of the uridine-containing medium had been taken up, 100,u1 portions of 0.1 M sucrose, 0.1 mM ammonium citrate in tap water were supplied for the remaining period of uptake. In labeling experiments with tritium, uridine-6- 3 H (New England Nuclear Corp., USA) in distilled water was substituted for the above medium, and plant segments were given tap water after the uptake of the initial solution. In all cases the entire radioactivity was taken up by the segments. Amounts of label and specific activities are given under results. At the end of the uptake period the two primary leaves of plant segments were harvested and used immediately or stored at - 25 DC before extraction of native RNA. Leaves of intact plants were used as carrier.

c) Chlorophyll determinations Twenty primary leaves were ground with 80 per cent acetone, calcium carbonate, and sand in a mortar. The homogenate was filtered and chlorophyll absorption was determined in a Zeiss PMQ spectrophotometer. The pigment concentration was calculated according to ARNON (1949):

(E~6~~m

X

8.0

+ Et4~~m X 20.2),ug

chlorophyll per mi.

d) Estimation of DNA and RNA per leaf Total DNA and RNA per leaf were determined according to the method described by SMILLIE and KROTKOV (1960). For unilJuminated leaves, the first step in the procedure was performed in green safelight. Steps no. 1-9 and 16-18 were done at 0-4 DC, the others at room temperature. 1. 50 leaves 18

Biochem. Physiol. Pflanzen, Bd. 162

K. S. ANDERSEN

248

were homogenized in a Sorvall Omni-mixer at speed setting 5 with 15 ml methanol for 15 min and centrifuged for 10 min at 3000 X g. The resulting pellet was treated as follows. At each step (except no. 14) the pellet was suspended in the appropriate solution by stirring and thereafter centrifuged for 10 min at 3000 X g, 2. 10 ml methanol, 3 -4. 10 m10.2 per cent formic acid in methanol, 5-6. 10 ml 5 per cent perchloric acid (PCA), 7 -8.10 ml 96 per cent ethanol, 9. 10 ml absolute ethanol, 10 -11. 10 ml of absolute ethanol: ethyl ether = 2 : 1, 12 -13. 10 ml boiling ethyl ether for 20 seconds, 14. the final pellet was air dried, Hi. 6 ml 0.3 M potassium hydroxide at 37 DC for 16-20 hours, cooling to 0 DC, 1 ml 70 per cent PCA added, suspension cooled again, 16. supernatant, 17 -18. 4 ml 5 per cent PCA and cooled, 19. 5 ml 5 percent PCA at 90°C for 15 min, 20-21. 4 ml 5 per cent PCA. The pooled supernatants from steps no. 15-18 were used for RNA determination in a Zeiss PMQ recording spectrophotometer and concentrations calculated according to SUNDERLAND and McLEISH (1961):

E~6~n:.m = 1.000 equals 32.5 /lg RNA per m!. The pooled supernatants from steps no. 19-21 were used for DNA determination, according to SUNDERLAND and McLEISH (1961): E~6~n:.m

=

1.000 equals 35,ug DNA per mI.

e) Phenol extraction of RNA from whole leaves Native RNA was extracted from whole leaves with phenol using a combination of methods described by FRAENKEL-CONRAT, SINGER and TSUGITA (1961), GIERER and SCHRAMM (1956), and SHEPHERD and PETERSEN (1962). Five g of deep-frozen leaves (labeled and unlabeled carrier leaves) were homogenized for 15 min at 0 DC at speed setting 3 in a Sorvall Omni-Mixer with 5 ml 0.1 M glycine buffer, pH 9.5, 20 ml water saturated phenol (EDTA-washed) and 0.6 ml 6 per cent bentonite. The mixture was centrifuged for 10 min at 3000 X g, the aqueous phase was removed, and the remaining material extracted with 5 ml of the glycine buffer. The second aqueous phase was pooled with the first, further extracted at 0-4 DC with 20 ml phenol, 0.2 ml 6 per cent bentonite, and then reextral ted with 15 ml phenol. The final aqueous phase was applied to a Sephadex G 25 column (volume 100 ml) equilibrated with 0.02 M TRIS/HCl, 0.15 M sodium chloride, pH 7.2. The elution was monitored at 260 nm by an LKB- Uvicord and -Recorder. The fractions from the first peak - containing the nucleic acids - were pooled and precipitated by adding one tenth volume of 3 J.{ sodium acetate and two volumes of 96 per cent ethanol. Precipitation was completed at - 25 DC. The precipitate was collected by centrifugation and dissolved in 0.02 M TRIS/HCl, pH 7.2. RNA solutions were stored at - 25 DC. RNA concentrations were calculated (KURLAND 1960) as E12~~m

=

1.000 equals 45.5,ug RNA per m!.

f) Chromatography of RNA on methylated albumin coated kieselguhr (MAK) column Methylated albumin was prepared as described by MANDELL and HERSHEY (1960). Columns (1 em X 25 cm) were packed according to SUEOKA and CHENG (1962). 2.7 mg of RNA was applied to a column and eluted with a 420 mllinear gradient from 0.3 to 1.3 M NaCI in 0.02 M TRIS/HCI, pH 7.2 at room temperature. The flow rate was 60 ml/hour and 5.5 ml fractions were collected. 1.5 ml aliquots of fractions were used to determine E 260 nm. The rest of each fraction was used for radioactivity determination after precipitation of the RNA for 30 min at 0 DC with

Ribosome Synthesis in Greening Primary Leaves of Bean Seedlings etc.

249

trichloroacetic acid (TOA) at a final concentration of 6 per cent. Bovine serum albumin at a final concentration of 0.006 per cent was used as coprecipitator. The precipitate was collected on Whatman glass paper filter (GF/O) and washed with 2 X 10 ml of cold 6 per cent TOA. 5.5ml of scintillation liquid (4 g of 2.5-diphenyloxazole + 0.05 g of 1.4- bis (2-( 4-methyl-5-phenyloxazolyl»-benzene per 1000 ml of toluene) was added and counts per min were determined in a Beckman 200 liquid scintillation spectrometer.

g) Ribosome isolation Ribosomes were isolated according to a modification of the procedure described by STUTZ and NOLL (1967). At 0-4 °0, 200-400 primary leaves (20-25 g) were ground with 80 ml of 0.1 M TRIS/HOl, 0.3 ~I sucrose, 5 mM magnesium acetate, pH 8.0 (tris-sucrose-Mg-buffer), and 100-120 g of sand in a mortar. The homogenate was filtered through 4 layers of cheese cloth and centrifuged for 20 min at 1000 X g. The sediment was called the chloroplast fraction and the supernatant was called the cytoplasmic fraction. The former contained intact chloroplasts, the latter conbined free cytoplasmic and chloroplastic ribosomes. To break the chloroplast envelopes in the chloroplast fraction, the pellet was suspended in 30 ml of 0.01 M TRIS/HOl, 5 mM magnesium acetate, pH 8.0 (tris-Mg-buffer). Ohloroplast fraction and cytoplasmic fraction were then centrifuged twice for 20 min at 20000 X g, and the resulting supernatants were centrifuged for 3 hours at 160000 X g in a Spin co L2 using the 65 rotor. The obtained ribosome pellets were washed four times with tris-Mg-buffer, then suspended over night in 0.5-1.0 ml of the same buffer, and centrifuged for 20 min at 20000 X g, to remove the remaining chlorophyll containing material. This resulted in suspensions called chloroplast fraction ribosomes and cytoplasmic fraction ribosomes. They were either used directly or stored for a few days at - 25 °0.

h) Sucrose gradient centrifugation 0.2 ml of ribosome suspension (1-2 mg) was layered onto a 5 ml 5-20 per cent (w/v in tris-Mg-buffer) linear sucrose gradient (BRITTEN and ROBERTS 1960) which had been kept over night at 0 °0. HO-labeled Escherichia coli 70S-ribosomes were used as reference. The gradients were centrifuged for 60 min at 39000 rpm in a Spinco L2 using the SW 50L rotor. Gradients were fractionated into 10 drop samples (0.2 ml per sample) by dripping out through a syringe needle. Samples were diluted with tris-Mg-buffer. Radioactivity was measured by adding 1 ml aliquots of samples to 10 ml portions of Bray's scintillation liquid (BRAY 1960) and counting in a Beckman 200 liquid scintillation spectrometer.

i) Electron microscopy Leaf discs (1.5 mm 2 ) were fixed in 4.2 per cent glutaraldehyde at 0 °0 for 2 hours, postfixed in osmium tetroxide at room temperature, dehydrated in a graded series of ethanol, and embedded in resin (SPURR 1969). Material to be used for radioautography was dehydrated in a graded series of ethanol, followed by a graded series of acetone in ethanol, and finally embedded in Durcopan (AOM, 1'luka). Sections were cut on an ultramicrotome and collected on copper grids or on formvar-carbon coated copper grids. Sections were poststained with uranyl acetate and lead citrate (REYXOLDS 1963) according to procedures described by PEASE (1964). Micrographs were taken with either a Zeiss EM 9A or a Siemens Elmiskop I electron microscope.

j) Radioautography Electron microscopic radioautography was performed as described by OARO and TUBERGEN (1964). Ilford L4 nuclear emulsion was used with Ilford 9041' safe light. With a loop of 0.25 mm thick copper thread - 35 mm in diamter - thin films of gelled emulsion were applied to sec18*

K. S.

250

ANDERSEN

tions. The coated sections were placed in light proof boxes at 0-4 DC for 48 days, and then developed and fixed at 20 DC in: for 5 min Kodak microdol X for 10 seconds 1 per cent acetic acid for 5 min Kodak quick finish fixer for 5 min running tap water for 5 min distilled water for 2 seconds 96 per cent ethanol For quantitative evaluation of radioautographs, cell and organelle areas were measured with a Haff 315 planimeter on micrographs of nonserial sections (final magnification 12900 times). Parts of cell organelles showing on micrographs were also measured and each was counted as one organelle. The cytoplasm from one cell was counted as one "organelle".

3. Results

a) Synthesis of equal amounts of chloroplastic and cytoplasmic ribosomes during chloroplast development The yield of phenol extracted RNA increased from 150 ttg in an 11 day old dark grown leaf to 250 ttg in such a leaf after 25 hours of light induced chloroplast development. If the dark growing leaf is supplied with 3H-6-uridine during a 24 hour period, the ribosomal-RNA's and the soluble-RNA's are labeled (fig.1a). Disre10

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Ribosome Synthesis in Greening Primary Leaves of Bean Seedlings etc.

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garding the small amount of label in DNA, approximately 80 per cent the label is found in the ribosomal-RNA, and the rest in RNA species eluting together with 4-5S-RNA. The same distribution of label between ribosomal and transfer type RNA is observed when the dark grown leaf after the 24 hour labeling period is illuminated for 25 hours (fig. 1 b). Percentage of label is determined by measuring the areas under the cts/min peaks in fig. 1 a & b. All extinction values were corrected to the values expected to be contributed by the labeled material, assuming identical patterns of RNA content in the labeled and in the carrier leaves. Under this assumption the specific activities of the different RNA's in the two labeling experiments are similar, indicating that a large part of the uridine supplied in the dark is available for incorporation into RNA synthesized during the following 25 hour light period. This might imply that the uridine pool-size in the dark grown leaf is big enough to supply the RNA synthesis during the 25 hour light development. 1.0..----..,.----~----,-___.,.._--~

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Fig. 1. MAK column chromatography of nucleic acids from leaves of plant segments, which had been fed for 24 hours in the dark with 100 ftc uridine-6- 3 H (9.34 CjmM) per plant. Extinction values were corrected for amount of carrier leaves added. a) 0 hours of light; labeled leaves were harvested after 24 hours of feeding; 2.24 per cent labeled leaves. b) 25 hours of light; after 24hours of feeding in the dark the plant segments were subjected to illumination for 25 hours and then harvested; 5.06 per cent labeled leaves. : E260nm, -------: ctsjmin.

K. S.

252

ANDERSEN

To determine the intracellular distribution of the label incorporated during the 45 hours of light induced chloroplast development, radio autographs were prepared from leaves of 16 day old dark grown seedlings supplied with 3H-6-uridine during the light period (Plate 13). The results of a quantitative evaluation is presented in fig. 2. A total of 1360 silver grains were counted over a leaf section area of 15285 ,um2. The background counts for a corresponding area were 33 grains. Section areas occupied by vacuoles and intercellular spaces comprised nearly 50 per cent of the total area but only 162 grains, i. e. 12 per cent, were located over these areas. Furthertil Itil


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Fig. 2. Distribu.tion of silver grains over and specific labeling in different parts of palisade and spongy parenchym cells as determined from radioautography of 0.06 ftm thick sections of leaves. Sixteen day old dark grown leaves were fed 1 ml tap water containing 100 ftc uridine-6- 3 H (6.55 CjmM) during 45 hours of illumination. Background: 33 grains over a control area of 15,285 ftm 2 • p: grains situated over the periphery of the vacuoles. c: grains over central parts of vacuoles. n: numbers of cell organelle sectors measured and counted.

Ribosome Synthesis in Greening Primary Leaves of Bean Seedlings etc.

253

more when the grains counted over vacuoles were grouped into centrally and perifer ally located ones, the majority fell into the latter group (fig. 2, lower left diagram). They are considered to result from isotope disintegrations in the cytoplasm or organelles bordering the vacuoles. We can therefore disregard the contribution of the vacuoles and intercellular spaces to the labeling pattern (upper left diagram of fig. 2). Of the remaining area, amounting to 8052 ,um2, about 45 per cent was occupied by chloroplasts, 45 per cent by cytoplasm and mitochondria, and approximately 8 per cent by nuclei and nucleoli, the percentages of grains observed over the different organelles were roughly proportional to the areas occupied by them. If the specific labeling of the different organelles is plotted (right diagram of fig. 2) it is obvious that the nucleoli exhibit a much higher degree of labeling per unit area than the chromatin, nucleoplasm, chloroplasts, mitochondria, and cytoplasm. Total RNA was extracted (SMILLIE and KROTKOV 1960) from the leaves of which discs had been used for radioautography and cts/min were determined. From this it was estimated that at least 0.005 nM of the tritiated uridine (6.55 C/mM) supplied had been incorporated into leaf RNA. With the assumptions that half the label would be associated with the chloroplasts, that a 16 day old dark grown leaf illuminated for 45 hours contains 2 X 108 chloroplasts (GYLDENHOLM 1968), and that approximately 50 ultramicrotome sections result from one chloroplast, it can be calculated that during 48 days of radioautographic exposure 0.25 nuclear disintegrations would be expected to happen per chloroplast section. Approximately half of these disintegrations would be registered as grains in the emulsion i. e. 0.12 grains per chloroplast section. The number of grains observed per chloroplast section were 0.5.

b) Chloroplast development and nucleic acid synthesis in leaves from intact plants and from plant segments

In the radioautographic analysis 16 day old dark grown plants were used so that the results could be rel1)ted to GYLDENHOLMS data (1968). Since younger leaves upon excision continue to synthesize RNA and protein longer than older leaves (W OLLGIEHN 1967) 11 day old dark grown seedlings were used in other experiments. If the seedlings were cut below the cotyledons, the primary leaves kept their RNA level, whereas removal of the cotyledons resulted in breakdown of leaf RNA and in slower greening. The rate of pigment and RNA synthesis of the plant segments could be further increased by supply of sucrose and ammonium citrate. Under these conditions, the primary leaves on the plant segments continued pigment and RNA accumulation for at least five hours at the levels of those of intact seedlings. Fig. 3 depicts the chlorophyll accumulation during chloroplast development. After the onset of illumination there is the usual 3 hour lag period before the rate of

K. S.

ANDERSEN

chlorophyll synthesis increases. Chlorophyll synthesis in the plant segments is maintained during 5 hours after excision (fig. 3, dashed lines). Chloroplast development was monitored in thin sections from leaves of intact seedlings and of plant segments excised for 5 hours. The stages analysed were: dark grown leaves and leaves illuminated for 5, 10, 15 and 25 hours. No effect of excision on development of chloroplast structures was detected by examining ultramicrotome sections from the intact seedlings and from the plant segments. Plates 14-17 show examples of micrographs from this analysis, demonstrating, the developmental stages reached after different illumination periods. Likewise, during 5 hours after excision the nucleic acid accumulation in the primary leaves of the plant segments was unimpaired at any of the developmental stages analysed (table 1). The DNA and RNA values at each stage reveal no significant differences between leaves from intact plants and from plant segments (table 1), nor are values from plant segments systematically lower or higher than values from intact plants. The RNA and the DNA values from each illumination period were pooled and plotted in fig. 4, in order to show the changes in nucleic acid content during greening of the primary leaves. The amounts of leaf DNA and RNA present increase significantly during chloroplast development. The data do not fit a linear increase, but both DNA and RNA values fit an exponential curve (cf. legend to fig. 4).

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255

Ribosome Synthesis in Greening Primary Leaves of Bean Seedlings etc. Table 1

Content of DNA (f-tg) and RNA (f-tg) in attached leaves and leaves of plant segments from 11 day old dark grown plants illuminated for various length of times Hours in light 0

RNA

0

DNA

5

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5

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15

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15

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20

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20

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25

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45

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384 442 44.8 52.2 410 462 46.2 52.2 449 507 48.7 56.7 609 631 74.0 65.8 715 449 74.9 74.9 575 611 89.3 73.5

341 299 30.4 30.1 417 400 50.8 49.4 436 445 82.6 53.9 715 566 62.0 61.3 484 449

603 571 81.9 79.1

432 404 42.4 41.3 358 344

279 230 28.7 24.5

388 378 46.2 45.2

319 280

462 445 34.1 45.5 492 593 52.5 54.3

845 728 100.8 105.7

The attached leaves (upper figures) and those from plant segments which had been excised during the last 5 hours (lower figures) before harvest and extraction, are from simultaneously grown seedlings, which were kept under otherwise identical conditions. DNA and RNA values are from the same preparations. A statistical test by paired comparison (FISHER 1948, p. 121) showed no significant difference between observations from attached leaves and leaves of plant segments, P-values ranging from 0.20 to 0.95.

c) Synthesis of chloroplastic and cytoplasmic ribosomes during chloroplast development Ribosomes isolated from the chloroplast fraction and the cytoplasmic fraction of dark grown leaves which had been fed for 24 hours with 3H-6-uridine in the dark and thereafter illuminated for 25 hours, distribute in sucrose gradients as shown in fig. 5. Fig. 5a pictures the values obtained by layering the total yield of ribosomes from the chloroplast fraction of 308 leaves onto the sucrose gradient. From the cytoplasmic fraction (fig. 5 b) an equal amount of ribosomes was layered onto the gradient. In order to depict the yield differences of the two fractions (fig. 5a vs. 5 b),

K. S.

256

ANDERSEN

the extinction and cts/min values were multiplied by a constant to give the values which would have resulted had it been possible to load the gradient with the total amount of ribosomes from the cytoplasmic fraction of 308 leaves. The yield of ribosomes from the chloroplast fraction is very low, due to lysis of most of the chloroplasts during the isolation procedure, and difficulties in artificially lysing the intact, purified chloroplasts. The ribosomes of the cytoplasmic fraction show a maximum in tube no. 14, whereas the chloroplastic ribosomes show a maximum in tube no. 17. The cytoplasmic ribosomes are heavily contaminated with chloroplastic ribosomes; 8oor-------~------~------~~------~--~

,ug RNA per leaf

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in;

The calculated regression lines are: For RNA: log y = 2.5604 ,For DNA: log y = 1.6026

+ 0.00641x + 0.00808x

Ribosome Synthesis in Greening Primary Leaves of Bean Seedlings etc.

257

with the procedure employed more chloroplastic ribosomes are in the cytoplasmic fraction than in the chloroplast fraction. To determine if the chloroplastic and cytoplasmic ribosomes which are made during chloroplast development are synthesized continuously or during a restricted period of organelle development, shorter labeling periods are necessary. This led to such low activities in the few ribosomes obtainable from the chloroplast fraction that only the cytoplasmic fractions were analysed (fig. 6). At five different stages of chloroplast development (v. WETTSTEIN and KAHN 1960), each of 10-18 plant segments werde fed 5 flc of uridine- 14 C-(U) during five hours. In this way ribosome labeling could be followed during greening. Micrographs demonstrate the stages of chloroplast development analysed: (a) proplastid with paracrystalline prolamellar

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Fig. 6. Sucrose gradient profiles of ribosomes from cytoplasmic fractions from plant segments fed for five hours with uridine-HC-(U) (495 mc/mM, 5 ftC per plant segment) in the dark and at different times after the onset of illumination. Unlabeled leaves of simultaneously grown and greened plants were added as carrier material. a) In dark: 18 plant segments were fed for 5 hours in the dark, 8.3 per cent labeled leaves. b) 5 hours in light: 15 plant segments were excised at time 0, and then illuminated and fed for 5 hours, 9.1 per cent labeled leaves. c)15hours in light: 10plants were illuminated for 10 hours, excised, and then fed in the light for 5 hours, 4.8 per cent labeled leaves. d) 25 hours in light: 10 plants were illuminated for 20 hours, excised, and then fed in the light for 5 hours, 6.3 per cent labeled leaves. e) 45 hours in light: 10 plants were illuminated for 40 hours, excised, and then fed in the light for 5 hours, 9.1 per cent labeled leaves. f) HC-Iabeled 70S ribosomes from E. coli. Data have been normalized to value of Emax. = 1, see text for explanation. - - - : E 260 nm, --------: cts/min.

Ribosome Synthesis in Greening Primary Leaves of Bean Seedlings etc.

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body developing in the dark (Plate 14), (b) plastid after 5 hours in light during which time dispersal of the prolamellar body into primary lamellar layers has taken place (Plate 15a), (c) plastid in early phase of grana development after 10 hours in light (Plate 15 b), (d) plastid after development for 15 hours in light, with chloroplastic ribosomes (Plate 16 a), (e) plastids after development for 25 hours in light, with advanced grana synthesis (Plate 16 b). In Plate 17 details of primary lamellar layers (a), developing grana (b), thylakoids with attached chloroplastic ribosomes (c, d) as described by FALK (1969) and mature grana (e) are depicted. Ribosomes isolated from the cytoplasmic fraction at five successive stages, distribute in sucrose gradients as shown in fig. 6. The data have been normalized in the following way: Assuming that ribosomes isolated from a leaf fed labeled uri dine and carrier ribosomes from intact plants of corresponding developmental stages distribute identically in sucrose gradients and ribosome isolation is equally efficient with the two kinds of leaves, all extinction values in each experiment were multiplied by the frequency of labeled leaves present in each case. In this way the extinction values for the ribosomes isolated from the uridine-fed leaves were determined. The data were then normalized to Emax. = 1.00, multiplying all extinction and ctsjmin values in each experiment by a constant. From fig. 6 a & b it is apparent that in the dark and during the first 5 hours of illumination (prolamellar body dispersal and primary lamellar layer formation in the plastids) the amount of labeling of both chloroplastic and cytoplasmic ribosomes is very low. At the time (fig. 6c) when grana are first being formed but before the grand pigment and grana synthesis period, chloroplastic and cytoplasmic ribosomes are very actively labeled. Later (fig. 6 d & e), during the grand period of grana synthesis and in mature chloroplasts, the amount of labeling of both types of ribosomes is again reduced, but remains higher than prior to the onset of chloroplast development. From the data presented in fig. 6 it is not possible to calculate precisely the specific activities (ctsjmin per mg ribosomes) of the chloroplastic and cytoplasmic ribosomes, as they were not resolved into two separate peaks in the gradients. A rough estimate can, however, be obtained from the gradient fractions no. 14 and no. 12, containing a preponderance of chloroplastic and cytoplasmic ribosomes, respectively. The relative specific activities of the ribosomes in fraction no. 14 vs. no. 12 are at 0 and 5 hours of light (fig. 6 a & b) the same, 0.5 vs. 0.6, indicating equal labeling of the two types of ribosomes. At 15, 25, and 45 hour light (fig. 6c, d & e) the values are 8.5 vs. 8.2, 5.0 vs. 4.6, and 7.4 vs. 7.0, respectively, indicating a slightly preferential labeling or synthesis of chloroplastic ribosomal RNA during grana formation. The observed shift of the maximum in extinction and ctsjmin values in the the 15 hour light experiment (fig. 6c), from fraction no. 12 to no. 14, i. e. to the maximum for 70S ribosomes (fig. 6f), may reflect a peak of chloroplastic ribosome

K. S.

260

ANDERSEN

synthesis during the 10 to 15 hour light period or a relatively low yield of cytoplasmic ribosomes in the preparation at this stage of development. The general conclusion which can be drawn from the results presented in fig. 6 is that both chloroplastic and cytoplasmic ribosomes can be labeled by feeding labeled uri dine at any of the periods analysed. This is interpreted to imply that both types of ribosomes are synthesized continuously during plastid development. 4. Discussion

BOARDMAN (1966) observed a higher content of RNA per leaf in primary leaves of green house grown bean plants than in leaves of dark grown seedlings. In the present studies a significant increase in RNA content - amounting to a doubling (fig. 4) from 350 flg to 650 flg per leaf - i. e. a 300 flg increase per leaf was found during a 45 hour illumination of primary leaves from 11 day old dark grown bean seedlings. At the time when considerable grana formation had taken place, i. e. after 25 hours of light, the increase amounted to about 50 per cent. This is consistent with results obtained after illumination of 16 day old dark grown bean seedlings (GYLDENHOLM 1968), although in these older leaves the increase in RNA per leaf is slower - from 400 flg to 500 flg per leaf, i. e. a 100 flg increase - during the first 45 hours of illumination. It can be concluded that in young bean leaves - 11 day dark grown - a 3 times more active RNA accumulation can be induced by light than in older bean leaves - 16 day dark grown. The results from MAK - column chromatography (fig. 1 a & b, extinction values) demonstrate that the RNA present both in the dark and in the light contains approximately 80 per cent ribosomalRNA, and 80 per cent of the labeled uridine incorporated into RNA during light induced plastid development is found in ribosomal-RNA (fig. 1 b). Although these results were obtained using 11 day old dark grown seedlings, they are likely to be valid also for the interpretation of the radioautographic results with 16 day old dark grown plants. Thus, the 45 per cent of label located in the chloroplasts and the 45 per cent in the cytoplasm (fig. 2) are taken to imply that about equal amounts of cytoplasmic and chloroplastic ribosomal-RNA are synthesized during the greening of the leaves. The high degree of labeling per unit area of nucleoli compared to other areas in the cells is an indication that precursor RNA for cytoplasmic ribosomal- RNS is synthesized or accumulated first in the nucleolus and from there transported into the cytoplasm. There is no indication of whether transport of chloroplastic ribosomal-RNA takes place from the nucleolus via the cytoplasm to chloroplasts or the chloroplastic ribosomal-RNA is synthesized in situ. To answer this, much shorter labeling periods are necessary, as used by GIBBS (1968) with

Ochromonas. According to INGLE (1968 b) a pair of cotyledons from 4 day old dark grown

Ribosome Synthesis in Greening Primary Leaves of Bean Seedlings etc.

261

radish seedlings increase over a 24 hour illumination period their cytoplasmic and chloroplastic ribosomal-RNA content with approximately 10,ug and 18,ug, respectively. In these cotyledons therefore, the induction of chloroplast development by light, stimulates chloroplastic ribosomal-RNA synthesis approximately 2 times more than cytoplasmic ribosomal-RNA synthesis. Such a specific stimulation of the synthesis of chloroplast associated RNA was not observed by GYLDENHOLM (1968) in the greening of 16 day old dark grown bean leaves, since half the total RNA increase could be ascribed to chloroplasts. In agreement with the latter results my radioautographic data (fig. 2) show that 45 per cent of the uri dine incorporated during greening were located in chloroplasts and 45 per cent in the cytoplasm. The rough estimates of relative specific activities of chloroplastic and cytoplasmic ribosomes isolated from the 11 day old dark grown leaves which were fed RNA precursor (fig. 6) indicate that in younger leaves the chloroplastic ribosomal-RNA synthesis is stimulated more than the cytoplasmic upon induction of greening. The DNA content in the primary leaves of 11 day old dark grown seedlings doubles during 45 hours of illumination (fig. 4), a result which is at variance with analyses of greening 16 day old dark grown plants (GYLDENHOLM 1968). The younger leaves also contained 4 times as much DNA as the older ones. Further studies will have to reveal, whether this difference is due to DNA degradation during ageing of the leaves growing in darkness or whether it has to be ascribed to difficulties in DNA determination. The significant doubling of DNA during chloroplast development is not necessarily related to mitotic divisions of the parenchyma cells. Active chloroplastic DNA synthesis occurs in rapidly expanding and greening leaves of light grown tobacco plants (W OLLGIEHN and MOTHES 1963, 1964) and a 5 to 10 fold chloroplast multiplication in the individual cells has been established for spinach leaves during development in light (POSSINGHAM and SAURER 1969). Assuming that approximately 20 per cent of leaf DNA can be ascribed to chloroplasts (GYLDENHOLM 1968) then a 5 to 10 fold chloroplast mUltiplication would account for the DNA doubling. Another argument against mitoses taking place is that the expansion of the dark grown leaf from approximately 1.5 cm 2 to approximately 4 cm 2 at 45 hours of illumination is readily accounted for by the development of big vacuoles and intercellular spaces (cf. fig. 2, lower left diagram) which are practically nonexisting in the dark grown leaf. In 16 day old dark grown seedlings DNA synthesis and chloroplast multiplication do not take place up to 45 hours of light (GYLDENHOLM 1968). The doubling of DNA during the first 45 hours of illumination in 11 day old seedlings may indicate that the phase of chloroplast multiplication and DNA synthesis in young seedlings already ensues during the first 45 hours of greening and plastid differentiation. Endopolyploidy of nuclei or chloroplasts and variable yields in DNA extraction from leaves of different age and developmental stage will have to be considered in further analyses of this phenomenon.

262

K. S. ANDERSEX

Results from labeling experiments with higher plants using RNA and DNA precursors, only give indications about rates of synthesis as long as nothing is known about nucleotide pool sizes in the leaf cells, and whether pool sizes vary with the developmental stages of the leaf. Acknowledgements The author is indebted to professor Dr. D. Y. WETTSTEIN for helpful advice and criticism and to Drs. T. NILSSON-TILLGREN and K. SICK for suggestions and encouragement. Thanks are due to Miss J. VERHEIN HANSEN and Mr. P. ERIKSEN for excellent technical assistance. This work has been financially supported by grants to Dr. D. v. WETTSTEIN from the United States Public Health Service, National Institute of Health (GM-10819), the Danish National Science Research Council and the Carlsberg Foundation.

Literature ARNON, D. I., Copper enzymes in isolated chloroplasts. Plant Physiol. 24, 1-15 (1949). BOARDMAN, N. K., Ribosome composition and chloroplast development in Phaseo/us vulgaris. Exp. Cell Res. 43, 474-482 (1966). BOARDMAN, N. K., FRANCKI, R. I. B., and WILDMAN, S. G., Protein synthesis by cell-free extracts from tobacco leaves III. Comparison of the properties and protein synthesizing activities of 70S chloroplast and 80S cytoplasmic ribosomes. J. :Mol. BioI. 17, 470-487 (1966). BRAWERMAN, G., The isolation of a specific species of ribosomes associated with chloroplast development in Euglena gracilis. Biochim. Biophys. Acta 72, 317 -331 (1963). - POGO, A. 0., and CHARGAFF, E., Induced formation of ribonucleic acids and plastid protein in Euglena gracilis. Ibid. 55, 326-334 (1962). BRAY, G. A., A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1, 279-285 (1960). BRITTEN, R. J., and ROBERTS, R. B., High-resolution density gradient sedimentation analysis. Science 131, 32-33 (1960). CARO, L. G., and TUBERGEN, R. P. v., High-resolution autoradiography. I. Methods. J. Cell BioI. 15, 173 -188 (1962). Cr,ARK, M. F., MATTHEWS, R. E. F., and RALPH, R. K., Ribosomes and polyribosomes in Brassica pekinensis. Biochim. Biophys. Acta 91, 289-304 (1964). EISENSTADT, J. M., and BRAWERMAN, G., The protein-synthesizing systems from the cytoplasm and the chloroplasts of Euglena gracilis. J. Mol. BioI. 10, 392-402 (1964). FAr,K, J., Rough thylakoids: polysomes attached to chloroplast membranes. J. Cell BioI. 42, 582-587 (1969). FISHER, R. A., Statistical methods for research workers. Edingburgh, p. 121 (1948). FRAENKEL-CONRAT, H., SINGER, B., and TSUGITA, A., Purification of viral RNA by means of bentonite. Virology 14, 54-58 (1961). GIBBS, S. P., Autoradiographic evidence for the in situ synthesis of chloroplast and mitochondrial RNA. J. Cell Sci. 3, 327 -340 (1968). GrERER, A., and SCHRAMM, G., Infectivity of ribonucleic acid from tobacco mosaic virus. Nature 177, 702 -703 (1956). GYLDENHOLM, A. 0., Macromolecular physiology of plastids V. On the nucleic acid metabolism during chloroplast development. Hereditas 59, 142-168 (1968).

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HOOBER, J. K., and BLOBEL, G., Characterization of the chloroplastic and cytoplasmic ribosomes of Chamydomonas reinhardi. J. Mol. BioI. 41, 121-138 (1969). INGLE, J., Synthesis and stability of chloroplast ribosomal-RNAs. Plant Physiol. 43, 1448-1454 (1968a). The effect of light and inhibitors on chloroplast and cytoplasmic RNA synthesis. Ibid. 43, 1850-1854 (1968b). WELLS, R., and POSSINGHAM, J. V., Origins of chloroplast ribosomal RNA. Abstract in "Autonomy and biogenesis of mitochondria and chloroplasts". Australian Acad. Sci., Canberra 8-13 December, p. 41, 1969. KURLAND, C. G., Molecular characterization of ribonucleic acid from Escherichia coli ribosomes I. Isolation and molecular weights. J. Mol. BioI. 2, 83-91 (1960). LOENING, U. E., and INGLE, J., Diversity of RNA components in green plant tissues. Nature 215, 363-367 (1967). LYTTLETON, J. W., Isolation of ribosomes from spinach chloroplasts. Exp. Cell Res. 26, 312 -317 (1962). MANDELL, J. D., and HERSHEY, A. D., A fractionating column for analysis of nucleic acids. Anal. Biochem. 1, 66 -77 (1960). PEASE, D. C., Histological techniques for electron microscopy. 2nd ed. Acad. Press, pp. 219 -225 and 234-236, 1964. POSSINGHAM, J. V., and SAUER, W., Changes in chloroplast number per cell during leaf development in spinach. Planta (Berl.) 86, 186-194 (1969). REYNOLDS, E. S., The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell BioI. 17, 208-212 (1963). SCOTT, N. S., and SMILLIE, R. M., Evidence for the direction of chloroplast ribosomal RNA synthesis by chloroplast DNA. Biochem. Biophys. Res. Comm. 28, 598-603 (1967). SHEPHERD, G. R., and PETERSEN, D. F., Separation of phenol and deoxyribonucleic acid by Sephadex gel filtration. J. Chromatog. 9, 445-448 (1962). SISSAKIAN, N. M., FILIPPOVICH, I. I., SVETAILO, E. N., and ALIYEV, K. A., On the proteinsynthesizing system of chloroplasts. Biochim. Biophys. Acta 95,474-485 (1965). SMILLIE, R. M., and KROTKOV, G., The estimation of nucleic acids in some algae and higher plants. Can. J. Botany 38, 31-49 (1960). - and SCOTT, N. S., Organelle biosynthesis: the chloroplast. In "Progress in molecular and subcellular biology". Springer-Verlag 1, 136-202 (1969). SPENCER, D., and WHITFELD, P. R., The nature of the ribonucleic acid of isolated chloroplasts. Arch. Biochem. Biophys. 117, 337 -346 (1966). SPURR, A. R., A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31- 43 (1969). STUTZ, E., and NOLL, H., Characterization of cytoplasmic and chloroplast polysomes in plants: evidence for three classes of ribosomal RNA in nature. Proc. Nat. Acad. Sci. (Wash.) 57, 774-781 (1967). SUEOKA, N., and CHENG, T.- Y., Fractionation of nucleic acids with the methylated albumin colum. J. Mol. BioI. 4, 161-172 (1962). SUNDERLAND, N., and McLEISH, J., Nucleic acid content and concentration in root cells of higher plants. Exp. Cell Res. 24, 541-554 (1961). TEWARI, K. K., and WILDMAN, S. G., Function of chloroplast DNA, I. Hybridization studies involving nuclear and chloroplast DNA with RNA from cytoplasmic (80S) and chloroplast (70S) ribosomes. Proc. Nat. Acad. Sci. (Wash.) 59, 569-576 (1968). 19

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WELLS, R., and BIRNSTIEL, M., Kinetic complexity of chloroplastal deoxyribonucleic acid and mitochondrial deoxyribonucleic acid from higher plants. Biochem. J. 112, 777 -786 (1969). WETTSTEIN, D. v., and KAHN, A., Macromolecular physiology of plastids. Proc. Eur. Reg. Conf. Electron Microscopy, Delft, vol. 2, 1051-1054 (1960). WOLLGIEHN, R., Nucleic acid and protein metabolism of excised leaves. Symp. Soc. for Exp. BioI. (Cambridge) 21, 231-246 (1967). and MOTHES, K., Uber DNA in den Chloroplast en von Nicotjana rus/ica. Naturwissenschaften 50, 95 - 96 (1963). and MOTHES, K., Uber die Incorporation von 3H-Thymidin in die Chloroplasten-DNS von Nicotiana rustica. Exp. Cell Res. 35, 52-57 (1964). ZELDIN, M. H., and SCHIFF, J. A., RNA metabolism during light-induced chloroplast development in Euglena. Plant Physiol. 42, 922-932 (1967). and SCHIFF, J. A., A comparison of light-dependent RNA metabolism in wild-type Euglena with that of mutants impaired for chloroplast development. Plant a (BerL) 81, 1-15 (1968). Author's address: Dr. KIRSTEN SCWAARD ANDERSEN, Institut of Genetics, University of Copenhagen, 0ster Farimagsgade 2 A, DK-1353 Copenhagen K (Denmark).

Explanation of Plates Plate 13 Radioautograph of a section through a parenchyma cell from a primary leaf of 16 day old dark grown bean seedling. The leaf was supplied with 50 flc uridine-6- 3 H (6 ..,}5 CjmM) during 45 hours of illumination. Silver grains are seen over sectioned nucleus (N), chloroplast (CH), and cytoplasm (C). X 36000. Plate 14 Section through a plastid from a primary leaf cell of an 11 day old dark grown bean seedling. The crystalline arrangement of the tubular membrams in the prolamellar body is apparent. CR: cytoplasm with ribosomes, CW: cell wall. X 85000. Plate 15 Plastids in sections from primary leaves of 11 day old dark grown bean seedlings illuminated for (a) 5 hours and (b) 10 hours. P: primary lamellar layers, CR: cytophtsm with ribosomes, G: grana, CW: cell wall. (a) x43000, (b) x43000. Plate 16 Plastids in sections from primary leaves of 11 day old dark grown bean planto illuminated for (a) 15 hours and (b) 25 hours. CR: cytoplasm with ribosomes, G: grana, CW: eell wall, CHR: chloroplastic ribosomes. (a) X 59000, (b) x47000. Plate 17 Sections of plastids from parenchyma cells in primary leaves of 11 day old dark grown bean seedlings in different stages of light induced chloroplast developmEnt. (a) 5 hours in light. P: primary lamellar layer, CR: cytoplasm with ribosomes. X 90000. (b) 10 hours in light. Theearly stage of grana formation. G: grana. X 90000. (c) and (d) 15 hours in light. CHR: thylakoid attached chloroplastic ribosomes, CR: cytoplasmic ribosomes. (c) X 90 000, (d) X 90000. (e) 25 hours in light. Late stage of grana formation. G: grana, CE: chloroplast envelope. X90000.