Electrophoretic study of plant histones: Comparison with vertebrate histones

BItCHIVES OF BIOCHEMISTRY .4ND BIOPHYSICS 161, 171-177 (1974)

Electrophoretic Study of Plant Histones: Comparison with

Vertebrate Histones

PAUL NADEAU, DOMINICK PALLOTTA AND JEAN-G. LAFONTAINE

Laboratoire de Biologie Cellulaire et Molkculaire, Ddpartement de Biologie, Universite Laval, Cite’ (Jniversitaire, Q&bee, Canada GlK ?‘Pd

Received July 23, 1973

The histones of seven plant species (barley, leek, onion, pea, radish, rye, and wheat) were isolated and compared to the histones of calf thymus and rat liver using electrophoresis on polyacrylamide-urea and polyacrylamide-SDS gels. It was found that the Fl histone of plants contains more subspecies and has generally higher molecular weights than their animal histone counterparts. Histones F3 of plants and animals have identical molecular weights and similar but not identical mobilities on polyacrylamide-urea gels. No histones were found in plants which have molecular weights and mobilities on polyacrylamide-urea gels which resemble the values for histones F2a2 and F2b of animals, but instead the series of histones observed differ from any of the animal histones. These plant histones may represent either substantially modi- fied forms of F2a2 and F2b, or else may be a different class of histone molecules unique to plants. Fractions F2al in plants and animals are identical in electrophoretic behavior, but seem to differ in degree of acetylation.

In recent years, the histones of a great variety of animal species have been studied and well characterized. The general con- elusion drawn from these investigations is that most organisms contain the same five major histone fractions. In the case of vertebrates, for instance, Panyim, Bilek, and Chalkley (1) found considerable ho- mogeneity among the histones, especially fractions F3 and F2a1, which showed no observable variation in their electrophoretic migration (2), Only minor differences were observed for the fractions F2b and F2a2, whereas Fl, the most lysine-rich histone, showed the most variation among the different species studied.

Less information is available concerning plant histones (3-S). Except for the histones of pea, few comparisons were made between plants and calf thymus. It was reported that striking similarities in the chromato- graphic and electrophoretic behavior, the amino acid composition, and the N-termi- nal amino acid exist between the histones of these t,wo species (3). The authors identi-

fied a nearly one-to-one correspondence between pea bud and calf thymus histones, and, in the case of F2a1, its primary structure was found to be almost identical in these two organisms (9). However, more recently, Mandal et al. (10) have reported some electrophoretic and immunological dissimilarities between animal (calf and rabbit thymus) and plant (coconut endo- sperm and pea bud) histones.

In view of these conflicting reports, we have undertaken a comparative study of histones in animal and plant species. For that purpose, the electrophoretic mobilities and the molecular weights of histones of several plants were determined, and comparisons were made with the histones of calf thymus and rat liver.

MATERIALS AND METHODS

Extractions were made from calf thymus, rat liver, barley (Hordeum vulgare), leek (A&urn porrum), onion (All&m cepa), pea (Pisum sati- vum), radish (Raphanus sativus), rye (Secale cereale), and wheat (Triticum vulgare). Calf thy-

171

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172 NADEAU, PALLOTTA, AND LAFONTAINE

mus was taken from recently slaughtered animals and frozen at -20°C until it was thawed for use. Rat livers were taken from freshly killed animals. Except for leak and onion, for which bulbs were used, the plants were grown for seven days in the dark, and the stems were cut and used immedi- ately.

All manipulations were carried out at 4°C. The tissues were homogenized in a grinding medium containing 0.25 M sucrose, 0.003 M CaClz , 0.1% Triton X-100, and 0.05 M Tris-HCI, pH 6.0. Calf thymus was homogenized in a Waring Blendor at low speed for 30 set (200 ml of grinding medium/l0 g of tissue) and the plant tissues at high speed for 60 set (250 ml of grinding medium/100 g of tissue). Rat liver was mechanically homogenized with six strokes of a Potter-Elvehjem tissue grinder (206 ml of grinding medium/l0 g of tissue). The homogenates were filtered through four layers of cheesecloth. For the plants, the juices were hand squeezed from the cheesecloth and then refiltered through eight layers of the same material. The homogenates were centrifuged at 1,ooOg for 10 min.

Following resuspension in the grinding medium and centrifugation under the same conditions, the pellets were suspended in 100 ml of 0.08 M NaCl, 0.02 M EDTA-NaOH, pH 6.3, stirred for W hr, and centrifuged at 1,500g for 10 min. The pellets were then suspended in 100 ml of 0.05 M Tris-HCl, pH 6.3, and centrifuged at 1,500g for 10 min. This wash- ing was repeated once. The pellets of crude chromatin were purified by solubilization in a 0.5 nuu EDTA solution adjusted to pH 7.5 with Tris (10 ml of solution for 100 g of starting plant tissue, 100 ml for 10 g of animal tissue) with 10-15 strokes of a mechanical homogenizer. The solutions were centrifuged at 2,000g for 10 min, and the pellets were discarded. For barley, rye, and wheat, the chromatin was precipitated by adding a measured volume of a 0.5 M NaCl solution to the supernatants to give a final concentration of 0.15 M. Precipitated chromatin was collected by centrifugation at 5,000g for 10 min and resolubilized in deionized water. In the case of leek, onion, pea, and radish, the chromatin was sheared for 5 min in the Virtis 45 homogenizer at full speed, and the insoluble residues were eliminated by centrifuging at 10,OOOg for 10 min.

In order to extract the histones, an equal volume of cold 0.8 N HZSOa was added to the chromatin solutions, and the mixtures were stirred for 30 min. The preparations were next centrifuged at 18,OQOg for 10 min. The resulting pellet was reex- tracted with cold 0.4 N H&S04 . Five volumes of 99% ethanol were added to the pooled supernatants, and the histones were precipitated for 3 days at -20°C. The Fl fraction was selectively extracted from rye chromatin solutions by an over-

night dialysis against a 0.5 M phosphate buffer, pH 5.5, containing 1.0 M urea (11). The extract was centrifuged at 45,000g for 45 min, and the super- natant was precipitated with 5 vol of absolute ethanol. Alternatively, rye chromatin was extracted with 0.5 M NaCl for 2 hr, centrifuged 6 hr at 18O,OOOg, and the histones precipitated from the supernatants (12). These proteins were recovered by centrifugation, washed with ethanol and ace- tone, and dried under vacuum.

Electrophoresis was carried out in 15% polyacrylamide 2.5 M urea vertical gels according to Panyim and Chalkley (13). Migration was either for 4 hr at 120 V (17 mA) for Q-cm gels or for 30 hr at 206 V (6 mA) for 24-cm gels. Gels stained with Amido Black were then destained in a Bio-Rad Model 170 Diffusion Destainer apparatus. Molecu- lar weights were determined on polyacrylamide- SDS’ gels according to Weber and Osborn (14). As was done by Panyim and Chalkley (15), the stand- ard curve was plotted using the known molecular weight of calf thymus histones. Gels were scanned on a Gilford recording spectrophotometer Model 240 and photographed on Kodak Panatomic-X film.

RESULTS

The five major histone fractions of calf thymus and rat liver were identified according to the nomenclature of Phillips and Johns (16), and, in the present work, were used as controls. Several methods have been used to selectively extract the most lysine- rich histones (Fl). Two of these methods were chosen and applied to the chromatin of rye. It can be seen in Fig. 1 that the three slowest migrating histones were extracted and correspond therefore to the Fl histone fraction. Since the isolation of the individual histone fractions is a very time consuming task, this extraction was not repeated for the other plant species. Instead, the assumption was made that the histones which have a mobility similar to the Fl histones of rye are also of the Fl type.

The Fl fraction of plants has a slower migration and generally a more complex electrophoretic pattern than the corresponding animal histones. Except for the radish, in which two bands are found, this fraction is con- sistently resolved into three bands (F11 , F~II , and FIIII), while, under the same

1 Abbreviations: SDS, sodium dodecyl sulfate; PH, plant-specific histones.

PLANT HISTONES 173

electrophoretic conditions, the Fl from animals is separated into either one or two bands (Figs. 24). All of the plant histone Fl subspecies have migrations which are different from those of their animal counterparts (Table I).

Electrophoresis of Fl histones on polyacrylamide-SDS gels resulted in the separa- tion of two to five bands for plants and two c>ach for calf thymus and rat liver (Fig. 5, Table II). The plant fractions generally have

FIG. 1. Polyacrylamide-urea gels electrophoresis of Fl histones from rye. (A) FI histones extracted with phosphate-urea, (B) Fl histones extracted with NaCl, (C) whole histones.

higher molecular weights (22,000-25,000) than those of corresponding animal proteins (21,000-22,000). However, except for the pea, there is an Fl subspecies found in all organisms studied which has approximately the same molecular weight (22,000).

Our results also show that, upon electrophoresis in polyacrylamide-urea gels, the seven plant species studied possess a histone fraction having a mobilit’y similar to the F3 fraction from animals. In some gels, a band, which disappears after reduction with 2- mercaptoethanol, is observed to migrate at half the speed of this histone. In view of the fact that only F3 fraction is capable of forming 2-mercaptoethanol-sensitive dimers, we have identified this plant histone as F3 (2).

Although the migration of F3 on polyacrylamide-urea gels is similar in plants and animals, statistical analysis (t test) reveals a significant difference (P < 0.05) between the migration ratios of plant and animal groups. This phenomenon is more easily observed in long gels in which the hist’ones were electrophoresed for 30 hr (Fig. 6). Such a difference is not observed within each group. However, the migration on polyacrylamide-SDS gels of all the F3 histones studied is found to be identical for both plants and animals.

One of the most striking observations

0 CT RL R 0 W

FIG. 2. Electrophoretic comparison of histones on polyacrylamide gels containing 2.5 M urea. Histones were extracted from calf thymus (CT), rat liver (RL), rye (R), barley (B), and wheat (W). Electrophoresis was performed at 120 V for 4 hr. Twenty micrograms of histones were loaded on each gel.


F2b 0

FIG. 3. Densitometer tracings of the polyacrylamide-urea gels illustrated in Fig. 1. The relative abundance of each fraction can be esti- mated from the scannings.

made during the course of the present investigation is the absence in the plants of both fractions F2b and F2a2 on polyacrylamide-urea gels. In the calf thymus and rat liver preparations, these histones migrate between fractions F3 and F2a1, but, in the plants, no bands are found in this region. Furthermore, while no animal histone fractions appear between F3 and Fl, we note the presence of two fractions in this region for rye, leek, pea, and wheat, and of three in the case of barley, onion, and radish. These histones are collectively called plant histones. The molecular weights of these histones range from 14,800 to 17,200 and are different from those obtained for the animal histones so far analyzed.

As for the fastest migrating plant histone, it is tentatively identified as F2al because its electrophoretic mobility is identical to that of F2al from both calf thymus and rat liver. This fraction is generally separated

FIG. 4. Densitometer tracings of polyacrylamide-urea gels. Histones were extracted from rye (R), pea (P), onion (0), leek (L), and radish (Ra). Electrophoresis was carried out at 120 V for 4 hr. Twenty micrograms were loaded on each gel.

into two bands upon electrophoresis in polyacrylamide-urea gels : the faster migrating band is known to contain nonacetylated molecules, while the slower one contains acetylated ones (17). In the two animals studied, the F2al histone is approximately equally distributed between the two bands. For the plant species examined, there is about five times more protein in the faster migrating band than in the slower. These results suggest that in the two animals studied, the percentage of acetylated histone is approximately equal to that of the nonacetylated form, and, in plants, approximately lo-20% of the F2al fraction is acetylated, while 80-90 % is unacetylated. A similar result was found for this histone isolated from pea bud (9).

DISCUSSION

The results obtained show striking similarities between the F2al and F3 fractions

PLANT HISTONES 175

F3 (oxidized) Fl

Fla Flb PH

F3 F2b F2a2 F2al

TABLE I

MIGRATION OF HISTONE FRACTIONS IN POLYACRYLAMIDE-UREA GELS RELATIVE TO THE MIGRATION OF F2al (MIGRATION RATIOS)”

Calf Rat liver Rye Wheat Barley Leek Onion Pea Radish thymus

0.399 0.391 0.457

0.489 0.499 0.495 0.505 0.509 0.502 0.503 0.512 0.526 0.519 0.516

0.535 0.544 0.567

0.577 0.573 0.588 0.587 0.594 0.650 0.664 0.666

0.724 0.737 0.736 0.733 0.736 0.736

0.746 0.744 0.769 0.764 0.766 0.775 0.763

0.782 0.792 0.792 0.810

0.799 0.805 0.818 0.819 0.814 0.817 0.825 0.829 0.823 0.841 0.836 0.880 0.868 1.000 1.000 1.000 1.000 1.060 1.090 1.000 1.000 1.000

a Electrophoresis was carried out as described in Materials and Methods. Depending upon the species studied, between 3 and 30 gels were measured for each value.

FIG. 5. Electrophoretic comparison of histones on polyacrylamide-SDS gels. Histones were extracted from calf thymus (CT), rat liver (RL), rye (R), barley (B), and wheat (W). Electrophoresis was carried out at 8 mA/gel for 5 h. Fifty micrograms of protein were loaded on each gel.

from plants and animals on the basis of a mobility on polyacrylamide-urea gels their electrophoretic behavior. However, no which is similar to that of animal histones. correspondence can be found between the However, there is one Fl subfraction in all plant-specific histones (PH) and the animal plants and animals studied which has a F2b and F2a2 fractions. As for the Fl frac- nearly constant molecular weight of 22,000 tion, t,here are no plant histones which have (Table II). These results suggest that this


TABLE II

DETERMINATION OF MOLECULAR WEIGHTS ON POLYACRYLAMIDE-SDS GELS”

Calf thymus Rat liver Rye Wheat Barley Leek Onion Pea

Fl 24,800 25,500 25,000 25,000 25,200 24,700 24,700

24,000 24,000 24,000 24,000 24,OCQ 23,500 23,000

22,000 22,000 22,000 22,000 22,000 22,800 22,300 21,000 21,000

PH 16,700 16,800 17,200 16,900 17,000 17,000 15,700 15,800 15,900 16,000 15,700 15,000 15,200 14,800 15,000

F3-F2b 14,000 14,000 14,000 14,000 14,100 14,200 14,000 13,800 F2a2 12,500 12,500 F2al 11,000 11,000 11,000 11,000 11,000 11,000 11,000 11,OQo

Q Molecular weights were determined as described in Materials and Methods. The number of different gels measured for each band varied between two and ten.

L

F3 i

7 I

FIG. 6. Densitometer tracings of 20-cm gels. Calf thymus (CT) and rye (R) were electrophoresed for 30 hr on polyacrylamide-urea gels. Twenty micrograms of histones were loaded on each gel. Note the difference in migration for F3 histones.

Fl histone has undergone changes in the primary structure which would modify its net charge, but not its molecular weight. A similar suggestion was made for the Fl histone of Drosophila (18, 19).

The small but significant difference observed in the migration ratios of F3 between plants and animals (Table I) is not due to a difference in their molecular weight. It is therefore possible that differences, present on polyacrylamide-urea electrophoresis and absent on polyacrylamide-SDS electrophoresis, are due to either small variations in the primary structure or in the extent of acetylation or phosphorylation of these proteins.

Further characterization of these histones will be necessary to clarify these points.

In all of the plants examined no histones are found which have mobilities on polyacrylamide-urea or on SDS gels comparable to the mobilities of histones F2b and F2a2 of animals. Instead a series of bands are observed which migrate between Fl and F3. We have chosen to call these histones PH and to temporarily distinguish them from histones F2a2 and F2b of animals. This does not imply that there are no homologous se- quences between these groups of histones. In fact, further work may prove that the homologies and physical properties of these histones are great enough to merit placing them in the same histone family. In this regard, it should be noted that, although the most lysine-rich histones of plants have different molecular weights and electrophoretic mobilities on polyacrylamide-urea gels than the corresponding animal histones, they have been placed in the same family.

The important point here is not the nomenclature assigned to these plant histones (PH), but the fact that they are different from the histones found in animals. Also, there is considerably less difference when histones from the plants are compared to each other than when they are compared to the histones of animals.

Previous studies, by ion-exchange chromatography and electrophoresis (4-6), led to the conclusion that pea bud and calf thymus

PLANT HISTONES 177

histones were similar and that a one-to-one correspondence could be established between the two kinds of histones. By ion-exchange chromatography, histones are separated due to differences in electrostatic in- teractions; it is possible that the charge differences between pea bud and calf thymus histones are two small to permit a separa- tion by this technique. Examining the same two materials by means of a higher resolu- tion electrophoretic technique, Panyim and Chalkley (3) found that the F2al and F3 fractions display similar migration, although distinct variations were observed to exist among the other fractions.

In accord with these authors, only fractions F2al and F3 are found to exhibit a similar electrophoretic behavior in the nine animal and plant species which we examined. Our findings that histones F2b and F2a2 seem to be absent from plants and that a group of proteins collectively called plant-specific histones (PH) are observed in these species cannot, at present, be easily accounted for. This result might have been interpreted as indicating that the plant histones (PH) play a key role in determining the quite charac- teristic reticulate organization of the interphase nuclei in many plants (20). However, our finding that radish, a plant species with interphase nuclei closely resembling nuclei of animal cells, possesses histones quite similar to the other plants studied indicates that this hypothesis is not valid.

The possibility must be considered that these plant histones (PH) play the same role in the nucleus as do the F2a2 and F2b fractions which are found in animals, but, due to evolutionary modifications, the primary structure of the two groups of histones is different. In this regard, it is interesting to note that, in the animals examined to date, the histone fractions F2a2 and F2b showed some variations in electrophoretic mobility and in molecular weight (2, 3, la), which suggest changes in the primary structure. These results indicate that significant change is permitted in these histones without loss of function.

ACKNOWLEDGMENTS

The authors thank Dr. D. P. Bloch for furnish- ing information concerning chromatin purifica-

tion, Dr. W. A. Anderson for his valuable advice and criticism during preparation of the manu- script, and Mr. S. Gugg for his excellent technical assistance. This investigation was supported by research grants from the Quebec Ministry of Education and the National Research Council of Canada. P. Nadeau was supported by a NRC post- graduate scholarship.

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Electrophoretic study of plant histones: Comparison with vertebrate histones

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