the deoxyribonucleic acid of broad bean chloroplasts

BIOCHIMICA ET BIOPHYSICA ACTA 417

BBA 8332

T H E DEOXYRIBONUCLEIC ACID OF BROAD BEAN CHLOROPLASTS

j . T. O. K I R K

Botany School, University o/ Ox]ovd, Ox]ord (Great Britain)

(Received May 24th, 1963)

SUMMARY

I. The question of whether higher-plant chloroplasts contain DNA has been investigated.

2. A method of obtaining highly purified broad bean chloroplasts by high- speed density-gradient centrifugation is described. The chloroplasts thus obtained have a DNA content, as measured by the diphenylamine reaction, of about o.15 % of the dry weight. About one-third of this DNA may be removed by repeated density- gradient centrifugation.

3. 66 % of the diphenylamine-positive material from the chloroplasts may be recovered after deproteinization and alcohol precipitation. Of this, 93 % is rendered soluble by DNAase (EC 3.1.4.5) treatment, indicating that it is DNA.

4. The chloroplast DNA.is much less readily extracted by HC104 than is nuclear DNA. Using gentle depurination followed by ion-exchange chromatography, the molar ratio of adenine to guanine has been found to be 1.67 for chloroplast DNA and 1.54 for nuclear DNA : this difference, although small, is statistically significant at the o.I % level. These results suggest that the DNA in the chloroplast preparations is not contaminating nuclear DNA.

INTRODUCTION

The manner in which chloroplasts are inherited in certain variegated plants has given rise to the belief that plastids are independent, self-reproducing entities, capable of undergoing mutation 1-3. It follows from this theory that chloroplasts should contain genetic information: they might therefore be expected to contain RNA or DNA or both. Many workers have claimed to have detected nucleic acids in chloroplasts cytologically 4-e, by autoradiography 7-9 and by chemical analysis of isolated chloroplasts 1°-15. .

Some of the cytological evidence has proved difficult to reproduce 16, but the recent work by RIS AND PLAUT 17, involving both light and electron microscopy, strongly suggests that in the alga, Chlamydomonas reinhardi, there is DNA in the chloroplast. The autoradiographic evidence consisted of the detection, within the chloroplast, of radioactivity from [3Hlthymidine ~-9. In the first two cases 7,s it was not shown ~hat the radioactivity was removable by DNAase (EC 3.1.4.5): however, WOLLGIEHN AND MOTClES 9 found that the radioactivity in chloroplasts of tobacco leaves could be removed with this enzyme. SAGAN AND SCHER 18 have reported the

Biochim. Biophys. Acta, 76 (1963) 417-424

418 J . T . O . KIRK

incorporation of I)NAase-removable label from ~3I-Ilthymidine into cytoplasmic structures in Euglena gracilis; it is not known whether this radioactivity was in the chloroplasts.

Isolated chloroplast preparations are probably always contaminated to some extent by other cell organelles, particularly by nuclei or nuclear fragments 1~. Reports that such preparations contain small amounts of nucleic acid do not, therefore, constitute good evidence that the chloroplasts themselves contain nucleic acid. One way of establishing that such nucleic acid was not due to contamination would be to demonstrate that it differed from the other cellular nucleic acids in its physical or chemical properties. An example of this is the report by BRAWERMAN 20 that the RNA of the ribosomes from Euglena chloroplasts has a different base ratio from the RNA of cytoplasmic ribosomes. Also LYTTLETON 21 found that ribosomes from spinach chloroplasts have a different sedimentation constant from that of cytoplasmic ribosomes. CHIBA AND SUGAHARA 22 reported that the DNA of highly purified tobacco and spinach chloroplasts is more difficult to extract with HC104 than the I)NA of crude chloroplast preparations. This suggests that the I )NA actually in the chloroplasts is more difficult to extract than the nuclear DNA which contaminates the crude preparations. Another possible example is the report by IWAMUR'A 23,24 that in Chlorella there are two kinds of DNA (one of which may be associated with chloroplasts) which differ in their base ratio and metabolic activity.

In this paper evidence is presented for the presence in broad bean chloroplasts of a type of DNA which differs in certain of its properties from nuclear DNA. Part of this work has already been published in abstract form 25.

METHODS

Preparation o/ chloroplasts and nuclei Broad bean plants were grown in a greenhouse and used when they were anything

between I I and 28 days old. Leaves (5o-15o g fresh wt.) were harvested, washed 3 times in tap water and twice in distilled water, and homogenized for 30-45 sec in a Townson and Mercer Top Drive Macerator, in 200 ml of a medium containing 0.5 M sucrose, 0.05 M Tris (pH 7-4) and I mM EI)TA (see ref. 26). The homogenate was strained through a double layer of muslin and centrifuged at 700 ×g for 2 min. The supernatant was centrifuged at IlOO ×g for 12 rain, yielding a pellet consisting mainly of chloroplasts with some nuclei. This pellet was resuspended in the homogenization medium and I-ml aliquots were placed on top of step-wise density gradients consisting of 0.5 ml of 35 % (w/v) sucrose layered on 1. 5 ml of 45 ~o (w/v) sucrose, which in turn was layered above I.O ml of 60 % (w/v) sucrose. The 35, 45 and 60 °/o (w/v) sucrose solutions were prepared by dissolving 2.31, 4.Ol and 6.89 g of sucrose, respectively, in IO ml of homogenization medium. The tubes were centrifuged at 76 ooo×g for 60 min in the swinging-bucket rotor of the MSE 4 ° ultracentrifuge.

The top layer and the 35 % layer were removed with a hypodermic syringe, the needle of which had a right-angle bend I cm from the tip, and were discarded. I ml of homogenization medium was gently run on to the 45 % layer and then removed and discarded. The 45 ~o layer, which contained practically all the chloroplasts, was removed, diluted with about 3 vol. of homogenization medium and centrifuged at 2000 x g for 30 min yielding a pellet of highly purified chloroplasts.


THE I ) N A OF BROAD BEAN CHLOROPLASTS 419

The clear supernatant which consisted of nuclei, as the nuclear fraction.

of the 6o % layer was removed leaving a pellet starch grains and a few chloroplasts. This was taken

Staining

The chloroplast and nuclear fractions were stained with 1.5 % acetoorcein and examined in the light microscope.

DNA and chlorophyll estimations

The chloroplast pellet was resuspended in distilled water. An aliquot was removed, diluted in 80 % acetone, allowed to stand in the dark for 30 min, and centrifuged. The absorbancies of the supernatant at 663 and 645 m# were measured, and the concentrations of chlorophylls a and b calculated, using the absorption coefficients given by ARNON 27.

To the remainder of the aqueous suspension of chloroplasts 4 vol. o,f ethanol were added. After 15 rain at room temperature the chloroplasts were centrifuged, extracted with absolute ethanol at 70-80 ° for IO min, and washed successively with methanol, 0.2 N IffC104 and 0.5 N HCIO a. The pellet was extracted twice with i-ml quantities of o.5 N HC104 for 15 min at 7 °0 (see ref. 28), and the extracts were combined. Two more extractions were then carried out with I-ml quantities of 2 N HC104 at 7 °0 for 15 rain and these extracts were combined, i-ml aliquots of the 0.5 N and 2 N extracts were used for the estimation of DNA by the diphenylamine procedure ~9. Sperm DNA (Nutritional Biochemicals Co.) was used as a standard. I t was found that DNA in 2 N HCI04 gave an Aeo 0 which was 28 % less than that given by the same amount of DNA in 0.5 N HC104. All the values of Aeoo for the 2 N HC104 extracts were therefore multiplied by 1.39 to make them equivalent to the values for the 0.5 N HC104 extracts.

I t was found that a third extraction of the chloroplasts with 2 N HCIO 4 yielded an amount of DNA which was not more than 8 % of the quanti ty already removed. Consequently only two treatments with 2 N HCIO a were used in these experiments.

Dry weight determination

Chloroplasts were resuspended in distilled water and an aliquot was taken for chlorophyll determination. HCIO 4 was added to a final concentration of 0.2 N. The suspension was allowed to stand for 30 min in an ice bath, centrifuged, washed once in distilled water, dried to constant weight in a vacuum desiccator and weighed. In this way the chlorophyll content was found to be 11.8-15.2 % of the dry weight.

DNAase treatment

The material to be treated was incubated for 19 h at 37 ° in 0.02 M phosphate buffer (pH 6.7) containing o.oi M MgSO4, in the presence or absence of DNAase (British Drug House Ltd.) at 20/~g/ml. HC104 was then added to a final concentration of 0.2 N and the tubes were left in an ice bath for 30 min. The precipitate was centrifuged, washed once in o.5 N HC104 and then DNA was extracted and estimated as usual.


420 J. T. O. KIRK

DNA preparation

DNA was extracted from chloroplasts or nuclei by the method of 1V[ARMUR 3°. After the first deproteinizatiou the nucleic acid was precipitated with 2 vol. of ethanol and centrifuged. The pellet was gently washed with 67 °/o (v/v) ethanol, with 80 % (v/v) ethanol, with absolute ethanol (twice) and with ether, and was then allowed to dry. The material was dissolved in 0.5 N NaOH and was incubated at 3 °0 for at least 16 h to hydrolyse the RNA. Glacial acetic acid was then added in amounts equivalent to 0.09 ml for every ml of solution, and the DNA was precipitated out by the addition of I vol. of ethanol. The precipitate was centrifuged and the pellet was washed gently with 67 % (v/v) ethanol, 80 % (v/v) ethanol, absolute ethanol (twice) and ether, and allowed to dry.

Hydrolysis and analysis o/ DNA

Acid hydrolysis of nucleic acids, even under relatively mild conditions (I N HC1 at IOO°), can bring about as much as 7 or 8 % deamination of adenine and guanine 31. Therefore'in an a t tempt to minimize degradation of the bases it was decided to hydrolyse the DNA at a pH which was only iust sufficiently acid to remove the purines. TAMM, HODES AND CHARGAFF s2 showed that when DNA was treated with o.I M glycine buffer (pH 2.8) at IOO ° for 60 min, the pyrimidine~ were not affected but the purines were quantitatively liberated.

In these experiments approx. 0.7 mg DNA was dissolved in 0.5 ml o.I M glycine- HC1 (pH 2.74) in a Pyrex test tube (io ×75 mm). The tube was placed in a boiling- water bath for 60 sec and then sealed tightly with a rubber bung. After a further 60 min in the water bath, the tube was removed and cooled. 0.24 ml of constant- boiling-point HC1 was then added to give a final concentration of 2 N. The tube was then centrifuged (although little or no turbidity appeared on addition of the acid) and the supernatant removed for analysis.

The amounts of adenine and guanine in the hydrolysate were determined by ion-exchange chromatography a3 on I)owex-5o (H + form, 200-400 mesh, 8 % cross- linked). The hydrolysate was allowed to run into a column of resin 36 mm high and IO mm in diameter, and was eluted with 2 N HC1. 3.5-ml fractions were collected and the absorbancies at appropriate wavelengths were measured by means of a Hilger Uvispek spectrophotometer.

Adenine (tubes 24-42 ) was well separated from other bases. The absorbancies at 275 and 31o m# were measured for each of the tubes in the adenine peak. The total amount of adenine was then calculated from the equation:

3.5 × ~ (A275--Aslo) /,moles adenine =

8.0

Absorbancies were measured at 275 m# rather than at the peak (262.5 m/~) because in 2 N HC1 adenine exists as a mixture of two ionic forms with different spectra. 275 m# is one of the isosbestic points for these two spectra and the millimolar extinction c.oefficient of adenine at this wavelength is 8.0 (measured on the curves given by BEAyEN, HOLIDAY AND JOHNSONa4).

The guanine peak extended from tubes 8 to 16. In tubes lO-16 only guanine was found and the absorbancies at 255, 265 and 31o m/~ were measured for each of theso

Biochirn. Biophys. Acta, 76 (1963) 417-424

THE DNA OF BROAD BEAN CHLOROPLASTS 421

tubes. Two different values for the amounts of guanine in these tubes were obtained from the equations:

3'5 X Y~(A,5 6 --Asto) /~moles guanine =

I 0 .0

3.5 x Y, (A2,s - - A,10) /~moles guanine ---- 7.2

and the mean value was taken. Guanine also exists as a mixture of two ionic forms in 2 N ttC1, the spectra of which have isosbestic points at 255 and 265 m#, with millimolar extinction coefficients of IO.O and 7.2, respectively (measured on the curves given by BEAVEN, HOLIDAY AND JOHNSONM). There was invariably a very small cytosine peak centred on tubes 6 and 7, indicating that hydrolysis had gone slightly farther than simple depurination. Consequently, tubes 8 and 9 contained trace amounts of cytosine as well as guanine. The amounts of guanine present in these two tubes were calculated from the equation:

[Y. (A., -- A,,o) -- 0.539 ".~ Y~ (A,5 -- A,1,)~ /,moles guanine 3.5 \ 6.i2 ]

and the value obtained was added to the mean value calculated for tubes lO-16. Of the remaining ultraviolet-absorbing material (which probably consisted

mainly of apurinic acids) about 75 ~/o was in tube I and the rest in tubes 2 and 3.

RESULTS

DNA content o/ puri/ied chloroplasts

The purified chloroplasts have a DNA content which is about I . I I °/o of the chlorophyll content (mean of a large number of experiments), although values as low as 0.54 ~o or as high as 1.73 % have been obtained. Thus the DNA content as a percentage of the dry weight of the chloroplast is in the region of o.15 ~o- No nuclei could be seen in chloroplast preparations stained with acetoorcein. However, it is not possible, on the basis of this staining procedure, to be absolutely certain that the preparations were uncontaminated by small fragments of nuclear material.

It is essential that the density-gradient centrifugation is carried out at high centrifugal fields. If low fields are used (2000 ×g) the chloroplast preparations have a much higher DNA content--about 2 ~/o of the dry weight. This is probably due to contamination by nuclear fragments.

E[/ect o/ [urther puri/ication In order to determine how firmly this DNA was bound, chloroplasts were carried

through one, two and three cycles of purification by density-gradient centrifugation. The results' in Table I show that during the second cycle of purification the DNA to chlorophyll ratio fell by 33 ~o; but that during the third cycle of purification it fell by only a further 6 %.

Increasing the duration of the density-gradient centrifugation led to somewhat variable results. In one experiment a density-gradient centrifugation lasting 5 h gave chloroplasts with a DNA to chlorophyll ratio identical with that of chloroplasts prepared in the normal way. On another occasion, increasing the length of density- gradient centrifugation from I h to 2 h brought about a 20 °/o fall in the DNA to chlorophyll ratio.


422 J . T . O . KIRK

T A B L E I

EFFECT OF REPEATED DENSITY-GRADIENT CENTRIFUGATION ON DNA CONTENT

P a r t of a pur i f ied ch lorop las t p r e p a r a t i o n was resuspended and car r ied t h rough a second dens i ty - g r ad i en t cen t r i fuga t ion . These ch lo rop las t s were h a r v e s t e d and a por t ion was subjec ted to a t h i rd d e n s i t y - g r a d i e n t cen t r i fuga t ion . The a m o u n t s of DNA and ch lo rophy l l in each of the

th ree ch lorop las t p r e p a r a t i o n s were measured .

Number of DNA Per cent purification - - (%) of first

cycles chlorophyll value

I 1.5o IOO 2 I.OO 67 3 o.91 61

Nature o/ diphenylamine-positive material I t was shown by BURTON 29 that the diphenylamine reaction is highly specific

for deoxyribose. Consequently, it has been assumed so far that the material measured by this procedure was, in fact, DNA. However, it was considered desirable to check this by seeing how much of the material could be removed by DNAase.

DNAase treatment of chloroplasts from which lipids had been extracted with hot ethanol, removed only 42 % of the diphenylamine-positive material. I t seemed possible that the chloroplast DNA might be protected from DNAase action as a result of being embedded within the chloroplast matrix. In order to liberate the I)NA from the surrounding material, a crude chloroplast nucleic acid preparation was made using the method of MARMUR s° as far as the first deproteinization and ethanol precipitation. This was incubated in the presence or absence of DNAase. ! t was found that the nucleic acid which had been treated with enzyme contained 93 % less diphenylamine-positive material than the control. However, in the control tube, only 66 % of the diphenylamine-positive material that had been present in the original chloroplasts was recovered. Thus of the amount of diphenylamine-positive material originally present in the chloroplasts, 62 ~o has been shown to be rendered soluble by DNAase.

Acid extractability o[ chloroplast DNA The results of CHIBA AND SUGAHARA 22 suggest that chloroplast DNA might be

unusually difficult to extract with HCIQ. Accordingly, it was decided to compare the ease of extraction of chloroplast DNA with that of nuclear DNA. The results presented in Table I I show that practically all the nuclear DNA is removed by 0.5 N HCI04 whereas to extract the bulk of the chloroplast DNA the concentration must be raised to 2 N.

TABLE II ACID EXTRACTABILITY OF CHLOROPLAST AND NUCLEAR DNA

Chloroplas t and nuc lear f rac t ions were t a k e n from the same dens i t y -g r ad i en t t ube and e x t r a c t e d success ive ly w i t h o. 5 N and 2 N HC104 in the u sua l way.

Material i~g DNA extracted Per cent I~g DNA Per cent o/ extracted of

by o. 5 N HCI04 total by 2 N HClOa total

Chloroplas t s 41.3 29 99.7 7 I Nuc le i 654 .0 97 21.6 3

B i o c h i m . B i o p h y s . A c t a , 76 (1963) 417-424

T H E DNA OF BROAD BEAN CHLOROPLASTS 423

Comparison o/base ratios o/chloroplast and nuclear DNA

If the DNA in the chloroplast preparations does not come from contaminating nuclear material then it should probably, although not necessarily, have a different base ratio from nuclear DNA. To investigate this the molar ratio of adenine to guanine was measured for 5 samples of chloroplast DNA and 5 samples of nuclear DNA. As may be seen in Table I I I the mean adenine to guanine ratio of chloroplast DNA is 8 % higher than that of nuclear DNA. Application of Student 's t test z5 indicates that this difference is significant at the o.I % level.

T A B L E I I I

THE A D E N I N E TO GUANINE RATIOS OF CHLOROPLAST AND NUCLEAR D N A

P o o l e d e s t i m a t e o f s t a n d a r d d e v i a t i o n s , s = o . o 3. D i f f e r e n c e b e t w e e n m e a n s = o . 1 3 . P r o b a b i l i t y t h a t t h e m e a n s a r e n o t d i f f e r e n t , _P < o . o o i .

Adenine (moles): guanine (moles)

Chloroplast DNA Nuclear DNA

Mean

L66 1.57 x .72 1.57 1.63 1.54 1.65 1.5 ° 1.67 1.54

1.67 1.54

DISCUSSION

It was shown by JAMES AND DAS 3e that ]ow-~peed density-gradient centrifugation of broad bean chloroplasts gave preparations which were free from mitochondrial contamination. The results presented in this paper indicate that this method can also be used to obtain chloroplasts essentially free of nuclear contamination, although much higher centrifugal fields are required to achieve this. The fact that further density-gradient purification can remove about a third of the chloroplast DNA might suggest that there is still a small amount of nuclear contamination. However, this loss could equally well be due to diffusion of DNA out of the chloroplasts, since chloroplasts isolated in aqueous media are extremely ]eaky~L Indeed, taking this possibility into account it seems not unlikely that the value obtained for the DNA content of the isolated chloroplasts, approx, o.I 5 °/o , is a minimum estimate; that the chloroplasts in the cell may contain much more DNA than this.

The results of DNAase treatment indicate that practically all the diphenylamine-positive material in a nucleic acid preparation from chloroplasts is DNA. The fact that only 66 % of the diphenylamine-positive material in the chloroplasts was recovered in the nucleic acid fraction was probably a result of losses in the course of the preparation, due to the very low concentration of DNA.

The DNA in the chloroplast preparations appears to differ from nuclear DNA in at least two respects. Firstly, it is much less readily extracted by HCIO 4 than is nuclear DNA: this may be due to the chloroplast DNA being protected by the surrounding chloroplast ]ipoprotein. Secondly, it has a higher molar ratio of adenine to guanine than nuclear DNA: the difference is small, but is highly significant,

Bio6him. Biophys. A¢ta, 76 (i963) 417-424

4 2 4 j . T . O . KIRK

statistically. If these chloroplast preparations still contained a small amount of nuclear DNA then the observed adenine to guanine ratio of the chloroplast DNA, 1.67, might be somewhat lower than that of pure chloroplast DNA.

The difference between the properties of the nuclear and the chloroplast DNA makes it unlikely that the DNA in the chloroplast preparations is from contaminating nuclear material.

ACKNOWLEDGEMENT

I am indebted to the Department of Scientific and Industrial Research for financial support.

REFERENCES

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Biochim. Biophys. Acta, 76 (i963) 417-424

the deoxyribonucleic acid of broad bean chloroplasts

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