- Email: [email protected]
Synthesis of ribonucleic acid in plants
Experimental
Cell Research 17, 227-236
SYNTHESIS I. DISTRIBUTION OF SUBCELLULAR
227
(1959)
OF RIBONUCLEIC
ACID
RIBONUCLEIC ACID AND COMPONENTS OF PEA
IN PLANTS OF PROTEIN EPICOTYLS’
AMONG
P. 0. P. TS’O and C. S. SAT0 Division
of Biology,
California
Institute
of Technology
Pasadena,
Calif,
U.S.A.
Received September 20, 1958
THE isolation
and partial characterization of microsomal nucleoprotein particles from pea seedlings has been reported from our laboratory [22]. The fact that cells contain such particles raises further questions, among which acid of the cell is contained in these are (1) how much of the total ribonucleic and (2) what is the origin of the microsomal RNA? In order to particles; answer these questions, it has been necessary to develop methods for quantitative separation of subcellular components which meet two requirements. These requirements are (1) the microsomal particle fraction must be free from contamination by either mitochondria or by the supernatant fraction; and (2) the cytoplasmic fraction must be free of nuclei. The basis for the second requirement is that in short period labeling experiments, the specific activity of nuclear RNA is much higher than that of the cytoplasmic fractions [19, 18, aoj. Previous fractionation procedures for plant materials ill, 15,16,21] have not satisfied these two requirements, since (1) the properties of the microsoma1 particles have not previously been known. Thus, the fate of the particles in the isolation procedtires and media previously employed have not been determined. High centrifugal forces are required to sediment the particles, and in several earlier studies an appropriate preparative ultracentrifuge was not available. In no earlier case has an analytical ultracentrifuge been employed to follow the procedure and therefore no check on completeness of separation of particles from supernatant was made. (2) No special effort has been made and no adequate assurance given that nuclei have been removed from the cytoplasmic fraction. In this communication we propose a fractionation procedure for subcellular components of plant tissues which satisfy the above specifications. This procedure has been adopted for studies of phosphate-32P incorporation into RNA 1 Report of work supported in part by grants No. Rg3977 and No. Rg5143 from the National Institutes of Health, United States Public Health Service. Experimental
Cell Research 17
P. 0. P. Ts’o and C. S. Sato of the various subcellular components [23]. The amounts of DNA, RNA, protein and of phosphorus in phosphoprotein in these components are reported for the case of pea seedling epicotyls. MATERIAL
AND
METHODS
Plant tissues.-Pea seedlings, Pisum sativum Alaska were grown in vermiculite in the dark for 7 days at 25°C. The apical 2.5 to 4.5 cm of the stems were collected. These are referred to below as shoots. In several experiments, only the apical 1.5-2 cm sections or only the 2 cm long subapical stem sections (below the apical section) were harvested. These are referred to as tip and stem sections respectively. The tissue was ground in a mortar at 2-4°C with 0.4 M sucrose solution (0.5 ml per gram of tissue). Before fractionation, the homogenate was filtered through cheese cloth by a press or through a fabric (Nu-fab, Essentials Products Inc., Los Angeles) with a suction flask aided by pressure applied to the top. The pH (6.4) and the electrolyte concentration (0.06 per cent) of these homogenates have been previously reported
[=I. Chemicals.-Ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) were obtained from California Foundation for Biochemical Research. Orcinol and diphenylamine were recrystallized twice before use. All other chemicals were of reagent grade. Equipment.-All centrifugations were done in the Spinco ultracentrifuge Model L with No. 40 rotor. The ultraviolet absorption measurements and calorimetric determinations were done in the Beckman spectrophotometer model DU. Analyses.-The modified method of Allen [l] was used for phosphorus determination. The orcinol method of Dische and Schwartz [6] and the diphenylamine method of Dische [l] were used for the determination of ribose and deoxyribose, respectively. The Nessler method [3] was used for total nitrogen. The same method, applied to samples from which nucleic acid had been removed, was used for protein nitrogdn. Amounts of DNA and RNA are expressed in micrograms per g of fresh weight.of tissue. The conversion of U.V. optical densities at 260 rnp to DNA and RNA are based on phosphorus; i.e., 11,000 O.D. for l(P) for RNA and 8800 O.D. for l(P) for DNA [17]. The value, thus, obtained in terms of phosphorus was then converted to micrograms of DNA and RNA by the factor 11, which is based on the fact that nucleic acids contain 9 per cent phosphorus 14, 141. RNA measurement by the orcinol method is based on a standard curve of yeast RNA of known weight and known phosphorus content dissolved in 1 N perchloric acid. Deoxyribose measurement of DNA by the diphenylamine method is based on a similar procedure. RESULTS Experiments
on fractionation
and extraction
Extraction of RNA by the method outlined above is 95 per cent complete. Thus, the tissue residue remaining after press-filtration contain about 5 per cent of the total RNA. Further squeezing of the residue with hydraulic press Experimental Cell Research 17
RNA and protein distribution
in pea epicotyls
229
or reextraction failed to yield more than 2-3 per cent RNA. It is concluded that the bulk of the RNA of the tissue is liberated from the tissue 1.0the pressfiltered homogenate. Analytical ultracentrifugation furnishes the basic information for the separation of the microsomal particles. These particles possess a sedimentation coefficient of 7.5s with aggregates of 110s and subunits of 40-60s [22]. The amounts of aggregates (9 per cent) and of subunits (5 per cent) is small in comparison to the amount of 75s particles. The homogenate was found to contain no detectable amount of components having sedimentation coefficients in the range of 400-120s or of 35 to 8s. Appropriate calculation based on this information shows that centrifugation at 110,000 X g for 90 minutes will quantitatively remove microsomal particles from the supernatant. Indeed, the centrifugal pattern and the RN,4 content of the supernatant (see below) so obtained, indicate the absence of microsomal particles in the supernatant. Similar calculations also show that little sedimentation of microsomal particles should occur by centrifugation at 42,000 X g for 12 minutes. Such centrifugation should therefore serve to free the microsomes from contamination by material of large sedimentation coefficients (8006100s) such as mitochondria. The high RNA to protein ratio (0,62, Table II) of ?he microsomal fraction, which agrees with that of the purified particles (0.65-0.68) [22 ], indicate the absence of mitochondria in this fraction. On the other hand, gentle washing of the mitochondrial pellet releases little RNA and indicates, therefore, the absence from this mitochondrial fraction of free microsomal particles. The supernatant and the mitochondrial fractions are, thus, welldefined. A matter of major concern is the separation of nuclei from cytoplasmic components. Filtered homogenates were centrifuged at 5000 x g for 6 minutes, 11 minutes, 15 minutes and 20 minutes, the pellet being designated as the nuclear fraction. The distribution of DNA between the pellets collected after various periods of centrifugation and the corresponding cytoplasmic fractions was followed by deoxyribose determination. At centrifugation periods of 11 to 15 minutes, the amount of DNA sedimented in the nuclear pellet attained a maximum and further centrifugation resulted in no increase in the amount of DNA contained in the pellet. It is concluded that centrifugation for 15 minutes at 5000 x g is sufficient to remove nuclei quantitatively. This process sediments 90-95 per cent of the total DX:,4 of the homogenates. The remaining few per cent of the DNA is found in the mitochondrial fraction (40,000 g for 12 minutes). No DNA could be detected in the microsomal or in the supernatant fraction. 16 ~ 593706
Experimental
Cell Research 17
P. 0. P. Ts’o and C. S. Sato
230
We propose a scheme based on the above experiments for separations of subcellular components into the four fractions: nuclear, mitchondrial, microsomal particles and supernatant. This scheme is presented in Fig. 1. Pea shoots, apical portion, 2.5 to 4.5 cm +0.5 ml 0.4 nf sucrose per gram of plant tissue. Ground using mortar and pestle at 2”-4°C filtered-fibrous part discarded. Centrifuge
homogenate
15 min., 8000 rpm I
I
pellet + 5 ml 0.2 M sucrose per tube centrifuge 10 min., 20,000 rpm I peilet (Nuclei fraction) transfer to conical centrifuge tube using 6 ml 0.5 N TCA
centrifuge
supernatant 12 min., 25,000 rpm I I
SUPERNATANT
I
(discard)
peilet 15 ml 0.2 M sucrose per tube centrifuge 12 min., 35,000 rpm I I
I
peilet (Mitochondria fraction) transfer to conical cenl trifuge tube using 6 ml 0.5 N TCA
Fig. l.-Scheme
I supernatant centrifuge 90 min. 40,000 rpm
supernatant (discard)
I I
I I
I pelilet (supernatant fraction) (Microsome fraction) add equal volume of transfer to conical cen1 N TCA in conical trifuge tube using 6 ml centrifuge tube 0.5 N TCA
for fractionation
of subcellular
components.
The next problem is the quantitative extraction of RNA and DNA. We have adopted the perchloric acid extraction procedure, essentially that of Ogur and Rosen [17], after unsuccessful trials by the enzymatic method [ll]. In the original procedure [17], 18 hours extraction in 1 N perchloric acid at low temperature is used to extract the RNA. In the present work, it has been found, however, that much longer extraction is required. TCA precipitates of the subcellular fractions were extracted with occasional stirring in 1 N perchloric acid at 2-4°C for various lengths of time. The residue was further extracted by hot 0.5 N perchloric acid. The amount of residual RNA extracted by the hot perchloric acid was calculated from phosphorus and U.V. absorption which was in excess of the amount of DNA as measured by deoxyribosc determination. Table I shows that RNA in nuclei and in the supernatant fraction was quantitatively extracted by cold 1 N perchloric acid in 48 hours, while 5-15 per cent of the RNA remained undissolved under the same condtions in the mitochondrial and microsome fractions. An extraction period of Experimental
Cell Research 17
RNA TABLE
and profein
disfribufion
231
in pea epicofyls
I. Extraction of RNA from all the cellular components by cold 1 N HClO, for various lengths of time followed by hot 0.5 N HClO,.
Time (hours) in cold HClO, ,..
Hot HCIOla
Cold HClO, 12
24
36
pg RNA/g
48
72
12
24 pg residual
fresh wt.
36
48
RNA/g
72
fresh wt.
Nuclei PO* 1J.V. Ribose
47.5 49.0 52.5
76.9 73.5 74.8
80.8 79.4 79.4
79.6
91.7 86.3 94.9
95.0 95.0 97.9
91.2 87.1 89.8
0.1
29.5 45.1
10.3 17.1
20.0 13.0
0.2
64.4 63.3
47.0 37.9
39.0 32.8
34.6 26.6
48.0 52.3
44.6 54.9
41.9 35.6
0.3 12.9
0.2 18.5
0.5 20.6
Mitochondria PO4 U.V. Ribose
78.3 83.4
108 103 114
Microsomes 352 387 373
PO, U.V. Ribose
442 450 456
421 453 426
453 494 429
477 534 472
143 165
24.4 27.0
Supernatant 8.4 27.0 9.7
PO, 1T.V. Hibose
10.0 34.9 14.4
18.8 48.0 23.1
15.0 45.0 22.0
0.5 19.1
a The contribution of DNA in the nuclear and mitochondrial fractions from the data. Other fractions contain no detectable amount of DNA.
have been subtracted
0.4
0.3 1Y ; 4 ‘ij .; 0.2 0
0.1
Fig. 2.-Ultraviolet absorption spectra of cold acid extracts (72 hours) of: 0, nuclei;, a microsomes; 0, mitochondria; and A, supernatant. 240 Wave
260 length
280
300
mp
Experimental
Cell Research 17
232
P. 0. P. Ts’o and C. S. Sato
72 hours in cold perchloric acid was, therefore, adopted. The U.V. absorption spectra of such extracts from nuclei, mitochondria, and microsomal particles indicate very little contamination by protein (Fig. 2). The supernatant fraction may be slightly contaminated by protein since its nucleic acid content as calculated from UV absorption is consistently higher than that based on ribose and phosphorus measurements (Table I). The extraction of DNA by the hot perchloric acid method has also been investigated. It has been found that two extractions (75”C, 20 minutes) are sufficient to remove all of the residual RNA (and the small amount of contaminating DNA in the mitochondrial fraction) from the cytoplasmic fractions. The nuclear fraction must, however, be extracted at least three times. In Table II a procedure for extraction and analysis is proposed for determination of RNA, DNA, protein, and residual protein phosphorus of the individual subcellular components. Amount and distribution of DNA, RNA, protein, and phosphorus of phosphoprotein among all the subcelMar components of pea shoots The amount and distribution of DNA, RNA, protein and phosphorus of the phosphoprotein of all of the subcellular fractions, based on three to seven replicates of typical experiments, are shown in Table III. II. Extraction and analytical prpcedure for RNA, DNA, protein, and phosphorus content in residual protein fractions (for 8 g of plant tissue).
TABLE
Pellets from all fractions, and precipitates from supernatants the following treatment: (1) (2) (3) (4) (5) (6) (7) (8) (9)
undergo
Left overnight in 0.5 N TCA at 0°C. Washed twice with 70 per cent EtOH (5 ml) at 2”-4°C. Washed twice with 0.1 per cent HClO, in 70 per cent EtOH (5 ml) at 2”-4°C. Washed twice with 1:2 Ether:EtOH (5 ml) at 50°C. Washed twice with 0.2 N HClO, (5 ml) at 2”-4°C. Left for 72 hrs. (with occasional stirring) in 4 ml 1.0 N HClO, at 2”-4°C; extracted. Washed twice with 1 ml 1.0 N HClO,. Aliquots of the 1 N HClO, extracts analyzed for phosphorus, ribose, and its U.V. spectrum, Residues of mitochondria, microsome, and supernatant fractions extracted twice with 4 ml and 2 ml 0.5 N HClO,, 20 min. at 75°C. (10) Residues of nuclei extracted twice with 3 ml and 2 ml 0.5 N HClO,, 20 min., at 75°C; extracted once more with 1 ml 0.5 N HClO,, 10 min., at 75°C. (11) The extracts of (9) and (10) analyzed for deoxyribose, phosphorus, and its U.V. spectrum. (12) The pellets digested and analyzed for total protein nitrogen and total phosphorus in the protein residue. Experimental
Cell Research 17
RNA
and protein
distribution
233
in pea epicotyls
The RNA contents of 2 other crops of plants were found to be about 20 per cent higher than indicated in Table III, but even in these cases the distribution of RNA was similar to that indicated in the table. Amount and distribution of RNA and protein and stem sections of pea shoots
in the tip
The plant material employed in Table III was the 3-4 cm portion of pea shoots, including the growing point, leaflets, and a portion of stem. It is of interest to separate the shoot further into the tip section (apical 1.5-2 cm) TABLE
III.
Amount
and distribution
of RNA and protein
@g/g fresh weight).
RNA Cold HClO,
Nuclei Mitochondria Microsome Supernatant
Protein
Hot HCIO,
1J.V.
Ribose
Phosphorus
Av.
Av.
Total
%
71.6 100.8 280.5 14.5
66.6 83.3 241.3 8.9
65.5 93.3 273.9 16.2
67.9 92.5 265.8 13.2
8.1 28.1 -
67.9 100.6 293.9 13.2
14.3 21.2 61.8 2.3
RNA/Prot.
%
0.125 0.278 0.62 0.012
25 16 19 40
DNA U.V. Nuclei Mitochondria Microsome Supernatant
TABLE
IV.
32.1
Comparison
Deoxyribose
Phosphorus
29.8 2.4 0 0
29.4
%
30.4 2.4
93 7
-
Phosphorus in phosphoprotein 2.8 1.4 .9 .93
of amount and distribution of RNA and protein fresh wt., stems and tips). RNA
Amount
Nuclei Mitochondria Microsome Supernatant
Av.
Protein Per cent
@g/g
RNA/Protein
Per cent
Per cent
Stems
Tips
Stems
Tips
Stems
Tips
Stems
Tips
37 60 94 11
150 224 736 57
18 30 47 5
13 19 63 5
28 17 13 41
23 11 19 46
0.062 0.17 0.35 0.026
0.13 0.40 0.77 0.05
Experimental
Cell Research 17
P. 0. P. Ts’o and C. S. Sato and the stem section (2 cm of stem below the tip section). The amount and the distribution of RNA and protein in these two sections are shown in Table IV. Dry weight, inorganic phosphate, content of the tissues
and orgmic
phosphate
Dry weights of the material were determined after heating the plant material at 100°C for 3 days. Dry weight as y0 of fresh weight was found to be 6.1 per cent for the whole apical portion of the seedlings, 8.5 per cent for the tip section and 5.5 per cent for the stem sections. The TCA-soluble phosphate pool in these tissues was measured in the following manner. Stem sections and tip sections were ground directly in the cold without addition of further liquid. The filtered homogenates were centrifuged at 100,000 x g for 24 hours to remove the heavy components. The clear supernatant was then precipitated with TCA at 0°C for 2 hours. The organic phosphate concentration of these undiluted cell saps determined by the method of Lowry and Lopez [la] is 2.0 x 10-a M and 2.4 x 10-s M for the stem sections and for the tip sections, respectively. The inorganic phosphate concentration of the sap of the stem is 7.9 X 10-S M and of the sap of the tip is 9.7 X 10-3 M.
DISCUSSION
The major conclusion from this work is that slightly more than 50-65 per cent of the cellular RNA is associated with the microsomal particles. They possess a high ratio of RNA to protein (0.4-0.75). Although the soluble RNA may have a particular and unique function [9], only 3 per cent of the total RNA is left in the supernatant after sedimentation of all particulate matter. It is because the properties of microsomal particles were not earlier known that this distribution of RNA in the plant cells was not earlier appreciated. Though the microsomal particles isolated by our procedure are relatively free of membraneous material, it is not definitely known that the endoplasmic recticulum is absent from these tissues. Such material has been reported to be present in the cells of the petioles of silver beet, roots of germinating wheat [lo], and corn roots [13], but to be absent in the meristematic cells of Vicia fabia [5]. The data presented above shows that an appreciable portion of the cellular RNA is present in nuclei and mitochondria. About lo-15 per cent of this Experimental
Cell Research 17
RNA and protein distribution
in pea epicotyls
235
RNA is present as a component with a sedimentation coefficient of 75s and which resembles microsomal particles. These nucleoprotein particles cannot he removed from nuclear and mitochondrial fractions by gentle washing but are released after homogenization together with freezing-thawing of these fractions. Further work is in progress regarding the origin and perhaps the attachment of these particles in the fractions of nuclei and mitochondria. The so-called “mitochondrial” and “nuclear” fractions of the present work are less definite. Since the nuclear fraction contains 93 per cent of the total cellular DNA, this fraction probably contains most of the nuclear fragments. The preparative procedures and chemical composition of nuclei and mitochondria have been adequately reviewed [2, 8, 191. Because of variations in source material, methods of analysis and even expression of results, we make no attempt here to analyze the earlier data critically or to compare them with our own. It has been reported [8, 11, 16, 211 that roughly 60-70 per cent of the RNA remains in the supernatant after the sedimentation of mitochondria in agreement with the findings in this paper. However, Martin and Morton report that in the silver beet, 60 per cent of the RN.A is removed with the nuclear and mitochondrial fractions [15]. If one takes the percentage of RNA and protein on a dry weight basis in mitochondria as 5-6 per cent and 30-40 per cent respectively [8], the RNA to protein ratio would be about 1 : 5 to 1 : 8. The ratio reported in this paper is about 1: 9 for the stem section, about 1: 2.5 for the tip section, and an average of 1: 4 for the mitochondria of the whole pea shoot. Two interesting comparisons may be made between the tip and stem sections as to amount and distribution of RNA and protein: (1) Though the ratio of dry to fresh weight of the tip section is only about 60 per cent higher than that of the stem section, the RNA content of the tip sections is about 4-7 times higher than that of the stem section. Furthermore, the RNA content per unit protein is twice as high in all components of the tip section as compared with those of the stem section. (2) In the older cells of the stem, more of the RNA and protein are sedimented with the nuclear and mitochondrial fractions. This is at the expense of the microsomal and supernatant fractions. Thus, the percentage of the total RNA and protein in the nuclear and mitochondrial fractions of the stem section is higher than in the same portions of the tip section. Similar changes in the proportions of microsomal and mitochondrial protein as between young and old cells are also found in corn roots [13].
Experimental
Cell Research 17
P. 0. P. Ts’o and C. S. Sato SUMMARY
A fractionation procedure is proposed for the subcellular components of the apical portion of the pea seedlings. This procedure emphasizes isolation of microsomal particles and separation of nuclei from cytoplasmic components. Amount and distribution of DNA, RNA, protein and phosphorus of phosphoprotein of these components from the homogenates of the apical portion of pea seedlings are reported. 50-60 per cent of the cellular RNA is found to be located in the microsomal fraction. This apical portion is further divided into the tip section and the stem section. Analyses of RNA, and protein of all the subcellular components from these two sections show that the components from the tip section are two times more enriched in RNA. REFERENCES R. J. L., Biochem. J. 34, 858 (1940). V. G., MIRSKY, A. E. and STERN, H., Advances in Enzymol. 16, 411 (1955). 2. ALLFREY, of BOXK and BENEDICT. In Practical Physiological Chemistry, by P. B. HAWK, B. 3. Formula L. OSER, and W. H. SUMMERSON. Ed. 12, p. 1230. Blakiston Co., 1951. Acids, p. 307. Edited by E. CHARGAFF and J. N. DAVISON, 4. CHARGAFF, E., in The Nucleic Academic Press, 1955. 5. CHAYEN, J. and JA&~N, F., Symposia Sot. Expff. Biof. 10, 134 (1957). 6. DISCHE, Z., in The Nucleic Acids, P. 301. Edited by E. CHARGAFF and J. N. DAVISON. Academic Press, New York, 1955: ibid. p. 287, 1955. 7. __ D. P., Znternat. Rev. Cytol. 4, 143 (1955). 8. HACKETT, M. B., ZAMICNICK, P. C. and STEPHENSEN, M. L., Biochim. et Biophys. Acfa 9. HOAGLAND, 24, 215 (1957). A. J., MARTIN, E. M. and MORTON, R. K., Biophys. Biochem. Cyfoi. 3, 61 (1957). 10. HOWE, 11. KMETEC, E. and NEWCOMBE, E. H., Am. J. Botany 43, 333 (1956). 12. LOWRY, -0. H. and LOPEZ, J. A., Biol. Chem. 16, 421 (1946): 13. LUND, H. A., VATTER, A. E. and HANSON, J. B., J. Biophys. Biochem. Cyfol. 4, 87 (1958). B., 9. Biophys. Biochem. Cyfol. 4, 373 (1958). 14. MAGASANIK, E. M. and MORTON, R. K., Biochem. J. 64, 221 (1956). 15. MARTIN. 16. MCCLENDON, J. H., Am. J. bofan. 39, 275 (1952). 17. OGUR, M. and ROSEN, G., Arch. Biochem. Biophys. 262 (1950). 18. SATO, C. S., PILCHER, J. R. and Ts’o, P. 0. P., Plant Physiof. 32, XII (Supplement) (1957). R. M. S., Infernat. Reu. Cyfol. 6, 383 (1957). 19. SIEBERT, S. and SMELLIE, Acids, p. 393. Edited by E. CHARGAFF and J. N. DAVISON. 20. SMELLIE, R. M. S., in The Nucleic Academic Press, New York, 1955. 21. STAFFORD, H. A., Physiof. Pfanfarum 4, 696 (1951). J., J. Biophys. Biochem. Cyfol. 2, 451 (1956). 22. Ts’o, P. 0. P., BONNER, J. and VINOGRAD, 23. Ts’o, P. 0. P. and SATO, C. S., Submitted for publication.
1. ALLEN,
Experimenfal
Cell Research 17
Cell Research 17, 227-236
SYNTHESIS I. DISTRIBUTION OF SUBCELLULAR
227
(1959)
OF RIBONUCLEIC
ACID
RIBONUCLEIC ACID AND COMPONENTS OF PEA
IN PLANTS OF PROTEIN EPICOTYLS’
AMONG
P. 0. P. TS’O and C. S. SAT0 Division
of Biology,
California
Institute
of Technology
Pasadena,
Calif,
U.S.A.
Received September 20, 1958
THE isolation
and partial characterization of microsomal nucleoprotein particles from pea seedlings has been reported from our laboratory [22]. The fact that cells contain such particles raises further questions, among which acid of the cell is contained in these are (1) how much of the total ribonucleic and (2) what is the origin of the microsomal RNA? In order to particles; answer these questions, it has been necessary to develop methods for quantitative separation of subcellular components which meet two requirements. These requirements are (1) the microsomal particle fraction must be free from contamination by either mitochondria or by the supernatant fraction; and (2) the cytoplasmic fraction must be free of nuclei. The basis for the second requirement is that in short period labeling experiments, the specific activity of nuclear RNA is much higher than that of the cytoplasmic fractions [19, 18, aoj. Previous fractionation procedures for plant materials ill, 15,16,21] have not satisfied these two requirements, since (1) the properties of the microsoma1 particles have not previously been known. Thus, the fate of the particles in the isolation procedtires and media previously employed have not been determined. High centrifugal forces are required to sediment the particles, and in several earlier studies an appropriate preparative ultracentrifuge was not available. In no earlier case has an analytical ultracentrifuge been employed to follow the procedure and therefore no check on completeness of separation of particles from supernatant was made. (2) No special effort has been made and no adequate assurance given that nuclei have been removed from the cytoplasmic fraction. In this communication we propose a fractionation procedure for subcellular components of plant tissues which satisfy the above specifications. This procedure has been adopted for studies of phosphate-32P incorporation into RNA 1 Report of work supported in part by grants No. Rg3977 and No. Rg5143 from the National Institutes of Health, United States Public Health Service. Experimental
Cell Research 17
P. 0. P. Ts’o and C. S. Sato of the various subcellular components [23]. The amounts of DNA, RNA, protein and of phosphorus in phosphoprotein in these components are reported for the case of pea seedling epicotyls. MATERIAL
AND
METHODS
Plant tissues.-Pea seedlings, Pisum sativum Alaska were grown in vermiculite in the dark for 7 days at 25°C. The apical 2.5 to 4.5 cm of the stems were collected. These are referred to below as shoots. In several experiments, only the apical 1.5-2 cm sections or only the 2 cm long subapical stem sections (below the apical section) were harvested. These are referred to as tip and stem sections respectively. The tissue was ground in a mortar at 2-4°C with 0.4 M sucrose solution (0.5 ml per gram of tissue). Before fractionation, the homogenate was filtered through cheese cloth by a press or through a fabric (Nu-fab, Essentials Products Inc., Los Angeles) with a suction flask aided by pressure applied to the top. The pH (6.4) and the electrolyte concentration (0.06 per cent) of these homogenates have been previously reported
[=I. Chemicals.-Ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) were obtained from California Foundation for Biochemical Research. Orcinol and diphenylamine were recrystallized twice before use. All other chemicals were of reagent grade. Equipment.-All centrifugations were done in the Spinco ultracentrifuge Model L with No. 40 rotor. The ultraviolet absorption measurements and calorimetric determinations were done in the Beckman spectrophotometer model DU. Analyses.-The modified method of Allen [l] was used for phosphorus determination. The orcinol method of Dische and Schwartz [6] and the diphenylamine method of Dische [l] were used for the determination of ribose and deoxyribose, respectively. The Nessler method [3] was used for total nitrogen. The same method, applied to samples from which nucleic acid had been removed, was used for protein nitrogdn. Amounts of DNA and RNA are expressed in micrograms per g of fresh weight.of tissue. The conversion of U.V. optical densities at 260 rnp to DNA and RNA are based on phosphorus; i.e., 11,000 O.D. for l(P) for RNA and 8800 O.D. for l(P) for DNA [17]. The value, thus, obtained in terms of phosphorus was then converted to micrograms of DNA and RNA by the factor 11, which is based on the fact that nucleic acids contain 9 per cent phosphorus 14, 141. RNA measurement by the orcinol method is based on a standard curve of yeast RNA of known weight and known phosphorus content dissolved in 1 N perchloric acid. Deoxyribose measurement of DNA by the diphenylamine method is based on a similar procedure. RESULTS Experiments
on fractionation
and extraction
Extraction of RNA by the method outlined above is 95 per cent complete. Thus, the tissue residue remaining after press-filtration contain about 5 per cent of the total RNA. Further squeezing of the residue with hydraulic press Experimental Cell Research 17
RNA and protein distribution
in pea epicotyls
229
or reextraction failed to yield more than 2-3 per cent RNA. It is concluded that the bulk of the RNA of the tissue is liberated from the tissue 1.0the pressfiltered homogenate. Analytical ultracentrifugation furnishes the basic information for the separation of the microsomal particles. These particles possess a sedimentation coefficient of 7.5s with aggregates of 110s and subunits of 40-60s [22]. The amounts of aggregates (9 per cent) and of subunits (5 per cent) is small in comparison to the amount of 75s particles. The homogenate was found to contain no detectable amount of components having sedimentation coefficients in the range of 400-120s or of 35 to 8s. Appropriate calculation based on this information shows that centrifugation at 110,000 X g for 90 minutes will quantitatively remove microsomal particles from the supernatant. Indeed, the centrifugal pattern and the RN,4 content of the supernatant (see below) so obtained, indicate the absence of microsomal particles in the supernatant. Similar calculations also show that little sedimentation of microsomal particles should occur by centrifugation at 42,000 X g for 12 minutes. Such centrifugation should therefore serve to free the microsomes from contamination by material of large sedimentation coefficients (8006100s) such as mitochondria. The high RNA to protein ratio (0,62, Table II) of ?he microsomal fraction, which agrees with that of the purified particles (0.65-0.68) [22 ], indicate the absence of mitochondria in this fraction. On the other hand, gentle washing of the mitochondrial pellet releases little RNA and indicates, therefore, the absence from this mitochondrial fraction of free microsomal particles. The supernatant and the mitochondrial fractions are, thus, welldefined. A matter of major concern is the separation of nuclei from cytoplasmic components. Filtered homogenates were centrifuged at 5000 x g for 6 minutes, 11 minutes, 15 minutes and 20 minutes, the pellet being designated as the nuclear fraction. The distribution of DNA between the pellets collected after various periods of centrifugation and the corresponding cytoplasmic fractions was followed by deoxyribose determination. At centrifugation periods of 11 to 15 minutes, the amount of DNA sedimented in the nuclear pellet attained a maximum and further centrifugation resulted in no increase in the amount of DNA contained in the pellet. It is concluded that centrifugation for 15 minutes at 5000 x g is sufficient to remove nuclei quantitatively. This process sediments 90-95 per cent of the total DX:,4 of the homogenates. The remaining few per cent of the DNA is found in the mitochondrial fraction (40,000 g for 12 minutes). No DNA could be detected in the microsomal or in the supernatant fraction. 16 ~ 593706
Experimental
Cell Research 17
P. 0. P. Ts’o and C. S. Sato
230
We propose a scheme based on the above experiments for separations of subcellular components into the four fractions: nuclear, mitchondrial, microsomal particles and supernatant. This scheme is presented in Fig. 1. Pea shoots, apical portion, 2.5 to 4.5 cm +0.5 ml 0.4 nf sucrose per gram of plant tissue. Ground using mortar and pestle at 2”-4°C filtered-fibrous part discarded. Centrifuge
homogenate
15 min., 8000 rpm I
I
pellet + 5 ml 0.2 M sucrose per tube centrifuge 10 min., 20,000 rpm I peilet (Nuclei fraction) transfer to conical centrifuge tube using 6 ml 0.5 N TCA
centrifuge
supernatant 12 min., 25,000 rpm I I
SUPERNATANT
I
(discard)
peilet 15 ml 0.2 M sucrose per tube centrifuge 12 min., 35,000 rpm I I
I
peilet (Mitochondria fraction) transfer to conical cenl trifuge tube using 6 ml 0.5 N TCA
Fig. l.-Scheme
I supernatant centrifuge 90 min. 40,000 rpm
supernatant (discard)
I I
I I
I pelilet (supernatant fraction) (Microsome fraction) add equal volume of transfer to conical cen1 N TCA in conical trifuge tube using 6 ml centrifuge tube 0.5 N TCA
for fractionation
of subcellular
components.
The next problem is the quantitative extraction of RNA and DNA. We have adopted the perchloric acid extraction procedure, essentially that of Ogur and Rosen [17], after unsuccessful trials by the enzymatic method [ll]. In the original procedure [17], 18 hours extraction in 1 N perchloric acid at low temperature is used to extract the RNA. In the present work, it has been found, however, that much longer extraction is required. TCA precipitates of the subcellular fractions were extracted with occasional stirring in 1 N perchloric acid at 2-4°C for various lengths of time. The residue was further extracted by hot 0.5 N perchloric acid. The amount of residual RNA extracted by the hot perchloric acid was calculated from phosphorus and U.V. absorption which was in excess of the amount of DNA as measured by deoxyribosc determination. Table I shows that RNA in nuclei and in the supernatant fraction was quantitatively extracted by cold 1 N perchloric acid in 48 hours, while 5-15 per cent of the RNA remained undissolved under the same condtions in the mitochondrial and microsome fractions. An extraction period of Experimental
Cell Research 17
RNA TABLE
and profein
disfribufion
231
in pea epicofyls
I. Extraction of RNA from all the cellular components by cold 1 N HClO, for various lengths of time followed by hot 0.5 N HClO,.
Time (hours) in cold HClO, ,..
Hot HCIOla
Cold HClO, 12
24
36
pg RNA/g
48
72
12
24 pg residual
fresh wt.
36
48
RNA/g
72
fresh wt.
Nuclei PO* 1J.V. Ribose
47.5 49.0 52.5
76.9 73.5 74.8
80.8 79.4 79.4
79.6
91.7 86.3 94.9
95.0 95.0 97.9
91.2 87.1 89.8
0.1
29.5 45.1
10.3 17.1
20.0 13.0
0.2
64.4 63.3
47.0 37.9
39.0 32.8
34.6 26.6
48.0 52.3
44.6 54.9
41.9 35.6
0.3 12.9
0.2 18.5
0.5 20.6
Mitochondria PO4 U.V. Ribose
78.3 83.4
108 103 114
Microsomes 352 387 373
PO, U.V. Ribose
442 450 456
421 453 426
453 494 429
477 534 472
143 165
24.4 27.0
Supernatant 8.4 27.0 9.7
PO, 1T.V. Hibose
10.0 34.9 14.4
18.8 48.0 23.1
15.0 45.0 22.0
0.5 19.1
a The contribution of DNA in the nuclear and mitochondrial fractions from the data. Other fractions contain no detectable amount of DNA.
have been subtracted
0.4
0.3 1Y ; 4 ‘ij .; 0.2 0
0.1
Fig. 2.-Ultraviolet absorption spectra of cold acid extracts (72 hours) of: 0, nuclei;, a microsomes; 0, mitochondria; and A, supernatant. 240 Wave
260 length
280
300
mp
Experimental
Cell Research 17
232
P. 0. P. Ts’o and C. S. Sato
72 hours in cold perchloric acid was, therefore, adopted. The U.V. absorption spectra of such extracts from nuclei, mitochondria, and microsomal particles indicate very little contamination by protein (Fig. 2). The supernatant fraction may be slightly contaminated by protein since its nucleic acid content as calculated from UV absorption is consistently higher than that based on ribose and phosphorus measurements (Table I). The extraction of DNA by the hot perchloric acid method has also been investigated. It has been found that two extractions (75”C, 20 minutes) are sufficient to remove all of the residual RNA (and the small amount of contaminating DNA in the mitochondrial fraction) from the cytoplasmic fractions. The nuclear fraction must, however, be extracted at least three times. In Table II a procedure for extraction and analysis is proposed for determination of RNA, DNA, protein, and residual protein phosphorus of the individual subcellular components. Amount and distribution of DNA, RNA, protein, and phosphorus of phosphoprotein among all the subcelMar components of pea shoots The amount and distribution of DNA, RNA, protein and phosphorus of the phosphoprotein of all of the subcellular fractions, based on three to seven replicates of typical experiments, are shown in Table III. II. Extraction and analytical prpcedure for RNA, DNA, protein, and phosphorus content in residual protein fractions (for 8 g of plant tissue).
TABLE
Pellets from all fractions, and precipitates from supernatants the following treatment: (1) (2) (3) (4) (5) (6) (7) (8) (9)
undergo
Left overnight in 0.5 N TCA at 0°C. Washed twice with 70 per cent EtOH (5 ml) at 2”-4°C. Washed twice with 0.1 per cent HClO, in 70 per cent EtOH (5 ml) at 2”-4°C. Washed twice with 1:2 Ether:EtOH (5 ml) at 50°C. Washed twice with 0.2 N HClO, (5 ml) at 2”-4°C. Left for 72 hrs. (with occasional stirring) in 4 ml 1.0 N HClO, at 2”-4°C; extracted. Washed twice with 1 ml 1.0 N HClO,. Aliquots of the 1 N HClO, extracts analyzed for phosphorus, ribose, and its U.V. spectrum, Residues of mitochondria, microsome, and supernatant fractions extracted twice with 4 ml and 2 ml 0.5 N HClO,, 20 min. at 75°C. (10) Residues of nuclei extracted twice with 3 ml and 2 ml 0.5 N HClO,, 20 min., at 75°C; extracted once more with 1 ml 0.5 N HClO,, 10 min., at 75°C. (11) The extracts of (9) and (10) analyzed for deoxyribose, phosphorus, and its U.V. spectrum. (12) The pellets digested and analyzed for total protein nitrogen and total phosphorus in the protein residue. Experimental
Cell Research 17
RNA
and protein
distribution
233
in pea epicotyls
The RNA contents of 2 other crops of plants were found to be about 20 per cent higher than indicated in Table III, but even in these cases the distribution of RNA was similar to that indicated in the table. Amount and distribution of RNA and protein and stem sections of pea shoots
in the tip
The plant material employed in Table III was the 3-4 cm portion of pea shoots, including the growing point, leaflets, and a portion of stem. It is of interest to separate the shoot further into the tip section (apical 1.5-2 cm) TABLE
III.
Amount
and distribution
of RNA and protein
@g/g fresh weight).
RNA Cold HClO,
Nuclei Mitochondria Microsome Supernatant
Protein
Hot HCIO,
1J.V.
Ribose
Phosphorus
Av.
Av.
Total
%
71.6 100.8 280.5 14.5
66.6 83.3 241.3 8.9
65.5 93.3 273.9 16.2
67.9 92.5 265.8 13.2
8.1 28.1 -
67.9 100.6 293.9 13.2
14.3 21.2 61.8 2.3
RNA/Prot.
%
0.125 0.278 0.62 0.012
25 16 19 40
DNA U.V. Nuclei Mitochondria Microsome Supernatant
TABLE
IV.
32.1
Comparison
Deoxyribose
Phosphorus
29.8 2.4 0 0
29.4
%
30.4 2.4
93 7
-
Phosphorus in phosphoprotein 2.8 1.4 .9 .93
of amount and distribution of RNA and protein fresh wt., stems and tips). RNA
Amount
Nuclei Mitochondria Microsome Supernatant
Av.
Protein Per cent
@g/g
RNA/Protein
Per cent
Per cent
Stems
Tips
Stems
Tips
Stems
Tips
Stems
Tips
37 60 94 11
150 224 736 57
18 30 47 5
13 19 63 5
28 17 13 41
23 11 19 46
0.062 0.17 0.35 0.026
0.13 0.40 0.77 0.05
Experimental
Cell Research 17
P. 0. P. Ts’o and C. S. Sato and the stem section (2 cm of stem below the tip section). The amount and the distribution of RNA and protein in these two sections are shown in Table IV. Dry weight, inorganic phosphate, content of the tissues
and orgmic
phosphate
Dry weights of the material were determined after heating the plant material at 100°C for 3 days. Dry weight as y0 of fresh weight was found to be 6.1 per cent for the whole apical portion of the seedlings, 8.5 per cent for the tip section and 5.5 per cent for the stem sections. The TCA-soluble phosphate pool in these tissues was measured in the following manner. Stem sections and tip sections were ground directly in the cold without addition of further liquid. The filtered homogenates were centrifuged at 100,000 x g for 24 hours to remove the heavy components. The clear supernatant was then precipitated with TCA at 0°C for 2 hours. The organic phosphate concentration of these undiluted cell saps determined by the method of Lowry and Lopez [la] is 2.0 x 10-a M and 2.4 x 10-s M for the stem sections and for the tip sections, respectively. The inorganic phosphate concentration of the sap of the stem is 7.9 X 10-S M and of the sap of the tip is 9.7 X 10-3 M.
DISCUSSION
The major conclusion from this work is that slightly more than 50-65 per cent of the cellular RNA is associated with the microsomal particles. They possess a high ratio of RNA to protein (0.4-0.75). Although the soluble RNA may have a particular and unique function [9], only 3 per cent of the total RNA is left in the supernatant after sedimentation of all particulate matter. It is because the properties of microsomal particles were not earlier known that this distribution of RNA in the plant cells was not earlier appreciated. Though the microsomal particles isolated by our procedure are relatively free of membraneous material, it is not definitely known that the endoplasmic recticulum is absent from these tissues. Such material has been reported to be present in the cells of the petioles of silver beet, roots of germinating wheat [lo], and corn roots [13], but to be absent in the meristematic cells of Vicia fabia [5]. The data presented above shows that an appreciable portion of the cellular RNA is present in nuclei and mitochondria. About lo-15 per cent of this Experimental
Cell Research 17
RNA and protein distribution
in pea epicotyls
235
RNA is present as a component with a sedimentation coefficient of 75s and which resembles microsomal particles. These nucleoprotein particles cannot he removed from nuclear and mitochondrial fractions by gentle washing but are released after homogenization together with freezing-thawing of these fractions. Further work is in progress regarding the origin and perhaps the attachment of these particles in the fractions of nuclei and mitochondria. The so-called “mitochondrial” and “nuclear” fractions of the present work are less definite. Since the nuclear fraction contains 93 per cent of the total cellular DNA, this fraction probably contains most of the nuclear fragments. The preparative procedures and chemical composition of nuclei and mitochondria have been adequately reviewed [2, 8, 191. Because of variations in source material, methods of analysis and even expression of results, we make no attempt here to analyze the earlier data critically or to compare them with our own. It has been reported [8, 11, 16, 211 that roughly 60-70 per cent of the RNA remains in the supernatant after the sedimentation of mitochondria in agreement with the findings in this paper. However, Martin and Morton report that in the silver beet, 60 per cent of the RN.A is removed with the nuclear and mitochondrial fractions [15]. If one takes the percentage of RNA and protein on a dry weight basis in mitochondria as 5-6 per cent and 30-40 per cent respectively [8], the RNA to protein ratio would be about 1 : 5 to 1 : 8. The ratio reported in this paper is about 1: 9 for the stem section, about 1: 2.5 for the tip section, and an average of 1: 4 for the mitochondria of the whole pea shoot. Two interesting comparisons may be made between the tip and stem sections as to amount and distribution of RNA and protein: (1) Though the ratio of dry to fresh weight of the tip section is only about 60 per cent higher than that of the stem section, the RNA content of the tip sections is about 4-7 times higher than that of the stem section. Furthermore, the RNA content per unit protein is twice as high in all components of the tip section as compared with those of the stem section. (2) In the older cells of the stem, more of the RNA and protein are sedimented with the nuclear and mitochondrial fractions. This is at the expense of the microsomal and supernatant fractions. Thus, the percentage of the total RNA and protein in the nuclear and mitochondrial fractions of the stem section is higher than in the same portions of the tip section. Similar changes in the proportions of microsomal and mitochondrial protein as between young and old cells are also found in corn roots [13].
Experimental
Cell Research 17
P. 0. P. Ts’o and C. S. Sato SUMMARY
A fractionation procedure is proposed for the subcellular components of the apical portion of the pea seedlings. This procedure emphasizes isolation of microsomal particles and separation of nuclei from cytoplasmic components. Amount and distribution of DNA, RNA, protein and phosphorus of phosphoprotein of these components from the homogenates of the apical portion of pea seedlings are reported. 50-60 per cent of the cellular RNA is found to be located in the microsomal fraction. This apical portion is further divided into the tip section and the stem section. Analyses of RNA, and protein of all the subcellular components from these two sections show that the components from the tip section are two times more enriched in RNA. REFERENCES R. J. L., Biochem. J. 34, 858 (1940). V. G., MIRSKY, A. E. and STERN, H., Advances in Enzymol. 16, 411 (1955). 2. ALLFREY, of BOXK and BENEDICT. In Practical Physiological Chemistry, by P. B. HAWK, B. 3. Formula L. OSER, and W. H. SUMMERSON. Ed. 12, p. 1230. Blakiston Co., 1951. Acids, p. 307. Edited by E. CHARGAFF and J. N. DAVISON, 4. CHARGAFF, E., in The Nucleic Academic Press, 1955. 5. CHAYEN, J. and JA&~N, F., Symposia Sot. Expff. Biof. 10, 134 (1957). 6. DISCHE, Z., in The Nucleic Acids, P. 301. Edited by E. CHARGAFF and J. N. DAVISON. Academic Press, New York, 1955: ibid. p. 287, 1955. 7. __ D. P., Znternat. Rev. Cytol. 4, 143 (1955). 8. HACKETT, M. B., ZAMICNICK, P. C. and STEPHENSEN, M. L., Biochim. et Biophys. Acfa 9. HOAGLAND, 24, 215 (1957). A. J., MARTIN, E. M. and MORTON, R. K., Biophys. Biochem. Cyfoi. 3, 61 (1957). 10. HOWE, 11. KMETEC, E. and NEWCOMBE, E. H., Am. J. Botany 43, 333 (1956). 12. LOWRY, -0. H. and LOPEZ, J. A., Biol. Chem. 16, 421 (1946): 13. LUND, H. A., VATTER, A. E. and HANSON, J. B., J. Biophys. Biochem. Cyfol. 4, 87 (1958). B., 9. Biophys. Biochem. Cyfol. 4, 373 (1958). 14. MAGASANIK, E. M. and MORTON, R. K., Biochem. J. 64, 221 (1956). 15. MARTIN. 16. MCCLENDON, J. H., Am. J. bofan. 39, 275 (1952). 17. OGUR, M. and ROSEN, G., Arch. Biochem. Biophys. 262 (1950). 18. SATO, C. S., PILCHER, J. R. and Ts’o, P. 0. P., Plant Physiof. 32, XII (Supplement) (1957). R. M. S., Infernat. Reu. Cyfol. 6, 383 (1957). 19. SIEBERT, S. and SMELLIE, Acids, p. 393. Edited by E. CHARGAFF and J. N. DAVISON. 20. SMELLIE, R. M. S., in The Nucleic Academic Press, New York, 1955. 21. STAFFORD, H. A., Physiof. Pfanfarum 4, 696 (1951). J., J. Biophys. Biochem. Cyfol. 2, 451 (1956). 22. Ts’o, P. 0. P., BONNER, J. and VINOGRAD, 23. Ts’o, P. 0. P. and SATO, C. S., Submitted for publication.
1. ALLEN,
Experimenfal
Cell Research 17