Biochemical Genetics, Vol. 13, Nos. 11/12, 1975
Polypeptide Composition of Fraction I Protein from Nicotiana glauca and from Cultivars of Nicotiana tabacum, Including a Male Sterile Line K. Chen, ~ S. D. Kung, 1'2 j . C. Gray, ~ and S. G. Wildman ~ Received 3 Feb. 1975--Final 13 May 1975
The polypeptide compositions of fraction I protein isolated from six collections of Nicotiana glauca and from ten eultivars of N. tabacum, as well as a polyploid series and a male sterile line, have been analyzed by isoelectric focusing in polyaerylamide gel slabs containing 8 M urea. Apart from the male sterile line, none of the plants showed any variation from the species pattern of polypeptides. Fraction I protein from the male sterile Burley 21 cultivar of N. tabacum contained the two small subunit polypeptides of N. tabacum and the three large subunit polypeptides of a n Australian species, probably N. megalosiphon, This indicates a changed chloroplast genome in the male sterile line in comparison to the normal fertile N. tabacum. K E Y W O R D S : fraction I protein; ribulose d i p h o s p h a t e carboxylase; Nicotiana; tobacco; m a l e sterility.
INTRODUCTION
Fraction I protein, the enzyme catalyzing carbon dioxide fixation in photosynthesis, is found in all green plants (Kawashima and Wildman, 1970). In the presence of denaturing agents such as urea or dodecylsulfate, the protein dissociates into its component polypeptides, which may be separated by S u p p o r t e d by R e s e a r c h G r a n t AI-00536 f r o m the N a t i o n a l Institutes of Health, U.S. Public H e a l t h Service, a n d by Contract AT(04-3)-34, Project 8, f r o m the Division o f Biology a n d Medicine, U.S. A t o m i c E n e r g y C o m m i s s i o n . 1 D e p a r t m e n t o f Biology a n d Molecular Biology Institute, University of California, Los Angeles, California. 2 Present address: D e p a r t m e n t o f Biological Sciences, University o f M a r y l a n d , Baltimore C o u n t y , Catonsville, M a r y l a n d . 771 © 1975 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher.
772
Chen, Kung, Gray, and Wildman
a variety of techniques. Separation on the basis of size produces two fractions of molecular weights 55,000 and 12,500 (Rutner and Lane, 1967; Kawashima, 1969; Rutner, 1970), currently known as the large and small subunits. However, recent separations by isoelectric focusing have shown a wider range of polypeptides. Fraction I protein from Nicotiana tabacurn was separated into three polypeptides with isoelectric points at pH 6.7-6.9 and two polypeptides with isoelectric points atpH 5.8-6.0. It was shown that the three polypeptides of higher isoelectric points had molecular weights of 55,000 and therefore comprised the large subunit, whereas the two polypeptides of lower isoelectric points had molecular weights of 12,500 and comprised the small subunit (Kung et al., 1974). Subsequent analysis of fraction I proteins isolated from other species of Nicotiana showed characteristic polypeptide patterns for all species. The marked differences between the polypeptide patterns of different species provide a wide variety of phenotypic markers which can be used in genetic and evolutionary studies in the genus Nicotiana (Sakano et al., 1974; Gray et al., 1974; Kung et al., 1975). The polypeptide patterns are especially useful for these studies because chloroplast DNA codes for the large subunit polypeptides (Chan and Wildman, 1972) and nuclear DNA codes for the small subunit polypeptides (Kawashima and Wildman, 1972). Examination of the polypeptide composition of a single protein can therefore provide information about the expression of both the chloroplast and nuclear genomes. The usefulness of this fraction I protein system depends on the absence of any wide variations in the polypeptide patterns within a given species. To examine the extent of intraspecies variation, we have analyzed fraction I protein from N. glauca, which occurs as a wild plant subject to the different environments of four continents, and from N. tabacum, the commerical tobacco plant which has been under constant manipulation by plant breeders for nearly 500 years. We have examined the polypeptide compositions of proteins isolated from the leaves of plants grown from six seed collections of N. gIauea and from ten cultivars of N. tabacum. Except for the fraction I protein of a male sterile line of N. tabaeum, there is no variation in polypeptide composition within these species. MATERIALS AND METHODS Seeds of N. glauea were collected from wild plants in Los Angeles, California; Guaymas, Mexico; Beersheba, Israel; Lake Naivasha, Kenya; and Adelaide, South Australia. Seeds of the cultivars of N. tabacum were obtained as follows: Turkish Samsun, BY 4, Burley 21, Ky 16, Ky 57, NC 95, Havana 38, and Maryland Mammoth were originally from the U.S. Department of Agriculture, Beltsville, Maryland; White Daruma and Green Daruma were from Hatano Tobacco Experiment Station, Japan Monopoly Corporation;
Polypeptides of Fraction I Protein from Nicotiana Cultivars
773
the isogenic Turkish Samsun was derived by doubling the chromosomes of a haploid plant produced by anther culture (Nitsch and Nitsch, 1969) and the white variegated mutant of Turkish Samsun arose as described by Wildman et al. (1973). Plants were grown under greenhouse conditions for 2-4 months after germination of the seeds, as described by Kawashima and Wildman (1972). Leaves of the polyploid series, based on Burley 21 and Ky 16, and of the male sterile Burley 21 were sent by airmail from Kentucky to Los Angeles. Transporting the leaves by this method does not result in any changes in the patterns obtained on isoelectric focusing. Crystalline fraction 1 protein was obtained from leaves (50 g wet weight) by a remarkably simple procedure (Chan et al., 1972). The leaves of nine plants grown in the same flat from a single seed collection were combined to make up each 50-g batch of leaves. The protein was recrystallized three times before analysis by isoelectric focusing, exactly as described by Kung et al. (1974). The crystalline fraction I proteins were dissolved in 8 Murea and S-carboxymethylated by reaction with iodoacetic acid before application to a prefocused 4.5~ polyacrylamide slab gel containing 1% Ampholine, pH 5-7, and 8 M urea. lsoelectric focusing was performed for 18 hr at 300 V and the polypeptide bands were visualized by staining with bromophenol blue. RESULTS Polypeptide Composition of Fraction I Protein from IV. glauca
The six seed collections of N. glauca, representing the four continents of America, Africa, Asia, and Australia, indicate the widespread distribution of this species in the world. However, as shown in Fig. 1, the polypeptide patterns of the fraction I proteins are similar among the six collections; each protein is made up of three large subunit polypeptides and a single small subunit polypeptide. There have therefore been no gross mutational changes in the fraction [ protein of N. glauca induced by changes in its environment as it spread around the world. Polypeptide Composition of Fraction I Protein from Cuitivars of IV. tabacum
The ten cultivars of N. tabacum analyzed are representative products of the intensive efforts of man to deliberately change the anatomy, cytology, physiology, disease resistance, climatic tolerance, and biochemical quality of the commercial product of a plant that has been under continuous cultivation in most regions of the world for several hundred years. However,
774
Chen, Kung, Gray, and Wildman
Fig. 1. Polypeptide composition of fraction I protein from N. g[auca collections. Polypeptides were separated by isoelectric focusing in polyacrylamide gel slabs containing 8 Murea and 1~ Ampholine,pH 5-7. Fraction I proteins applied at 20/~g per sample. From left to right, N. glauca collected from (1) California; (2) Australia; (3) Israel; (4) Kenya; (5) Mexico; (6) California. Abbreviations: L, large subunit polypeptides; S, small subunit polypeptides.
as shown in Figs. 2 and 3, the polypeptide patterns of the ten cultivars are identical; the protein is composed of three large subunit polypeptides and two small subunit polypeptides. We also analyzed fraction I protein isolated from the white tissue of a variegated mutant of Turkish Samsun and from an isogenic Turkish Samsun, and in each case the polypeptide pattern obtained was identical to the patterns of the other cultivars.
Polypeptide Composition of Fraction I Protein from a Polyploid Series of PC. tabacum
The polyploid series, containing diploid, triploid, and tetraploid plants, was based on the Burley 21 and Ky 16 cultivars. As shown in Fig. 3, there were no differences in the polypeptide patterns between the individual samples and the patterns were similar to those of the other cultivars. This indicates that gene dosage does not affect the polypeptide composition of fraction I protein.
Polypeptides of Fraction I Protein from Nicotiana Cultivars
775
L
Fig. 2. Polypeptide composition of fraction I protein from cultivars of N. tabacum. Polypeptide chains were separated by isoelectric focusing as described in the caption of Fig. 1. Cultivars, from left to right: (1) Turkish Samsun, (2) BY 4, (3) Ky 57, (4) Maryland Mammoth, (5) NC 95, (6) White Daruma, (7) GreenDaruma, (8) Havana 38.
Polypeptide Composition of Fraction I Protein from a Male Sterile N. tabacum
The male sterile line analyzed was a Burley 21 cultivar. The flowers were characterized by the complete absence of anthers but could be fertilized by pollen from normal fertile Burley 21 plants. The analysis of the fraction I protein is shown in Fig. 3, and it can be seen that the polypeptide pattern is different from that of the polyploid series and other eultivars of N. tabacum. The difference is in the position of the large subunit polypeptides, whereas the small subunit polypeptides are identical to those of the other cultivars. The large subunit polypeptides are similar to those of the Australian species of Ni¢otiana (see Sakano et al., 1974). It was deduced from the isoelectric focusing patterns that the male sterile line had been derived from the hybridization of an Australian species as the female parent and N. tabacum as the source of pollen for the fertilization. Repeated backcrossing and introduction of N. tabaeum nuclear genes for the small subunit of fraction I protein would result in the elimination of the Australian-type small subunit polypeptides and the substitution of the two small subunit polypeptides of N. tabacum. The backcrossing, however, would not affect the Australian
776
Chen, Kung, Gray, and Wildman
Fig. 3. Polypeptide composition of fraction I protein from a polyploid series and from a male sterile line of N. tabacum. Polypeptide chains were separated by isoelectric focusing as described in the caption of Fig. 1. Samples, from left to right: (1) diploid (Burley 21), (2) diploid (Ky 16), (3) diploid (F~ hybrid, Burley 21 x Ky 16), (4) triploid (Ky 16 4n x Ky 16 2n), (5) triploid (Burley 21 4n x Ky 16 2n), (6) triploid (Ky 16 4n x Burley 21 2n), (7) tetraploid (Burley 21 4n), (8) male sterile Burley 21. cytoplasm, in particular the chloroplast genes for the large subunit polypeptides, and would give rise to a tobacco line with the nuclear gene characteristics of N. tabacurn. DISCUSSION The present study indicates that in N. gIauca and in normal true breeding N. tabacurn there is no variation in the polypeptide patterns of fraction I protein obtained by isoelectric focusing. Although this finding was not entirely unexpected because the protein was crystallized from a homogenate
Polypeptides of Fraction I Protein from Nicotiana Cultivars
777
of several plants within the species, it was slightly surprising because the seed collections were from plant material that was highly inbred due to selfpollination over many generations and this would be expected to perpetuate any slight variation in the polypeptide pattern. These analyses, therefore, increase our confidence that intraspecies variation will not obscure the results obtained in genetic and evolutionary studies due to interspecies differences. The most surprising result in the present study was the polypeptide composition of the male sterile line of iV. tabacum. However, a search of the breeding record of this particular cultivar (S. J. Sheen, personal communication) revealed that an Australian species, either N. suaveolens or N. megalosiphon, was the female parent in the original cross with N. tabacum, as predicted from the polypeptide pattern. Both Australian species have large subunit polypeptides which correspond to those of the male sterile line and both species have three small subunit polypeptides, none of which corresponds to the two small subunit polypeptides ofN. tabaeum, indicating that the small subunit polypeptides of the male sterile line were indeed derived from the nuclear genome of AT. tabacum. Backcrossing the male sterile cultivar with the two Australian species produced seed only when N. megMosiphon was the pollen source, possibly suggestingthat N, megalosiphon, and not N. suaveolens, was the species involved in the origin of the male sterile line. There are two important consequences of this analysis of fraction I protein from the male sterile Burley 21 cultivar of N. tabacum. First, it demonstrates that in male sterile lines, which are of great advantage to plant breeders because they allow controlled pollination without hand emasculation, the chloroplast genome cannot be altered by conventional breeding techniques. Second, it indicates that in studies on the physiological basis of male sterility greater emphasis ought to be placed on the effects of the chloroplast genome, especially as the mitochondrial genome has been traditionally regarded as the source of the cytoplasmic factor in male sterility.
ACKNOWLEDGMENTS We thank Dr. N. Kawashima of the Hatano Tobacco Experiment Station, Japan Monopoly Corporation, for the gift of seeds, Dr. S. J. Sheen of the Department of Plant Pathology, University of Kentucky, Lexington, for seeds and for shipping the leaves of the polyploid series and the male sterile line of N. tabacum, and Professor C. A. Schroeder of this institution for numerous collections of seeds of N. glauca in various parts of the world. Seeds of the isogenic Turkish Samsun cultivar of N. tabacum were provided by the late J. P. Nitsch.
778
Chen, Kung, Gray, and Wildman REFERENCES
Chan, P. H., and Wildman, S. G. (1972). Chloroplast DNA codes for the primary structure of the large subunit of fraction I protein. Biochim. Biophys. Acta 277:677. Chan, P. H., Sakano, K., Singh, S., and Wildman, S. G. (1972). Crystalline fraction I protein: Preparation in large yield. Science 176:1145. Gray, J. C., Kung, S. D., Wildman, S. G., and Sheen, S. J. (1974). Origin of Nicotiana tabacum L. detected by polypeptide composition of fraction [ protein. Nature 252:226. Kawashima, N. (1969). Comparative studies on fraction I protein from spinach and tobacco leaves. Plant Cell Physiol. 10:31. Kawashima, N., and Wildman, S. G. (1970). Fraction I protein. Ann. Rev. Plant Physiol. 21:325. Kawashima, N., and Wildman, S. G. (1972). Studies on fraction [ protein IV: Mode of inheritance of primary structure in relation to whether chloroplast or nuclear DNA contains the code for a chloroplast protein. Biochim. Biophys. Acta 262:42. Kung, S. D., Sakano, K., and Wildman, S. G. (1974). Multiple peptide composition of the large and small subunits of Nicotiana tabacum fraction I protein ascertained by fingerprinting and electro-focusing. Biochim. Biophys. Acta 365:138. Kung, S. D., Gray, J. C., Wildman S. G., and Carlson, P. S. (1975). Polypeptide composition of fraction I protein from parasexual hybrid plants in the genus Nicotiana. Science 187:353. Nitsch, J. P., and Nitsch, C. (1969). Haploid plants from pollen grains. Science 163:85. Rutner, A. C. (1970). Estimation of the molecular weight of ribulose diphosphate carboxylase sub-units. Biochem. Biophys. Res. Comrnun. 39:923. Rutner, A. C., and Lane, M. D. (1967). Nonidentical subunits of ribulose diphosphate carboxylase. Biochem. Biophys. Res. Commun. 28:531. Sakano, K., Kung, S. D., and Wildman, S. G. (1974). Identification of several chloroplast DNA genes which code for the large subunit of Nicotiana fraction I proteins. Mol. Gen. Genet. 130:91. Wildman, S. G., Lu-Liao, C., and Wong-Staal, F. (1973), Maternal inheritance, cytology and macromolecular composition of defective chloroplasts in a variegated mutant of Nicotiana tabacum. Planta 113:293.
Polypeptide Composition of Fraction I Protein from Nicotiana glauca and from Cultivars of Nicotiana tabacum, Including a Male Sterile Line K. Chen, ~ S. D. Kung, 1'2 j . C. Gray, ~ and S. G. Wildman ~ Received 3 Feb. 1975--Final 13 May 1975
The polypeptide compositions of fraction I protein isolated from six collections of Nicotiana glauca and from ten eultivars of N. tabacum, as well as a polyploid series and a male sterile line, have been analyzed by isoelectric focusing in polyaerylamide gel slabs containing 8 M urea. Apart from the male sterile line, none of the plants showed any variation from the species pattern of polypeptides. Fraction I protein from the male sterile Burley 21 cultivar of N. tabacum contained the two small subunit polypeptides of N. tabacum and the three large subunit polypeptides of a n Australian species, probably N. megalosiphon, This indicates a changed chloroplast genome in the male sterile line in comparison to the normal fertile N. tabacum. K E Y W O R D S : fraction I protein; ribulose d i p h o s p h a t e carboxylase; Nicotiana; tobacco; m a l e sterility.
INTRODUCTION
Fraction I protein, the enzyme catalyzing carbon dioxide fixation in photosynthesis, is found in all green plants (Kawashima and Wildman, 1970). In the presence of denaturing agents such as urea or dodecylsulfate, the protein dissociates into its component polypeptides, which may be separated by S u p p o r t e d by R e s e a r c h G r a n t AI-00536 f r o m the N a t i o n a l Institutes of Health, U.S. Public H e a l t h Service, a n d by Contract AT(04-3)-34, Project 8, f r o m the Division o f Biology a n d Medicine, U.S. A t o m i c E n e r g y C o m m i s s i o n . 1 D e p a r t m e n t o f Biology a n d Molecular Biology Institute, University of California, Los Angeles, California. 2 Present address: D e p a r t m e n t o f Biological Sciences, University o f M a r y l a n d , Baltimore C o u n t y , Catonsville, M a r y l a n d . 771 © 1975 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher.
772
Chen, Kung, Gray, and Wildman
a variety of techniques. Separation on the basis of size produces two fractions of molecular weights 55,000 and 12,500 (Rutner and Lane, 1967; Kawashima, 1969; Rutner, 1970), currently known as the large and small subunits. However, recent separations by isoelectric focusing have shown a wider range of polypeptides. Fraction I protein from Nicotiana tabacurn was separated into three polypeptides with isoelectric points at pH 6.7-6.9 and two polypeptides with isoelectric points atpH 5.8-6.0. It was shown that the three polypeptides of higher isoelectric points had molecular weights of 55,000 and therefore comprised the large subunit, whereas the two polypeptides of lower isoelectric points had molecular weights of 12,500 and comprised the small subunit (Kung et al., 1974). Subsequent analysis of fraction I proteins isolated from other species of Nicotiana showed characteristic polypeptide patterns for all species. The marked differences between the polypeptide patterns of different species provide a wide variety of phenotypic markers which can be used in genetic and evolutionary studies in the genus Nicotiana (Sakano et al., 1974; Gray et al., 1974; Kung et al., 1975). The polypeptide patterns are especially useful for these studies because chloroplast DNA codes for the large subunit polypeptides (Chan and Wildman, 1972) and nuclear DNA codes for the small subunit polypeptides (Kawashima and Wildman, 1972). Examination of the polypeptide composition of a single protein can therefore provide information about the expression of both the chloroplast and nuclear genomes. The usefulness of this fraction I protein system depends on the absence of any wide variations in the polypeptide patterns within a given species. To examine the extent of intraspecies variation, we have analyzed fraction I protein from N. glauca, which occurs as a wild plant subject to the different environments of four continents, and from N. tabacum, the commerical tobacco plant which has been under constant manipulation by plant breeders for nearly 500 years. We have examined the polypeptide compositions of proteins isolated from the leaves of plants grown from six seed collections of N. gIauea and from ten cultivars of N. tabacum. Except for the fraction I protein of a male sterile line of N. tabaeum, there is no variation in polypeptide composition within these species. MATERIALS AND METHODS Seeds of N. glauea were collected from wild plants in Los Angeles, California; Guaymas, Mexico; Beersheba, Israel; Lake Naivasha, Kenya; and Adelaide, South Australia. Seeds of the cultivars of N. tabacum were obtained as follows: Turkish Samsun, BY 4, Burley 21, Ky 16, Ky 57, NC 95, Havana 38, and Maryland Mammoth were originally from the U.S. Department of Agriculture, Beltsville, Maryland; White Daruma and Green Daruma were from Hatano Tobacco Experiment Station, Japan Monopoly Corporation;
Polypeptides of Fraction I Protein from Nicotiana Cultivars
773
the isogenic Turkish Samsun was derived by doubling the chromosomes of a haploid plant produced by anther culture (Nitsch and Nitsch, 1969) and the white variegated mutant of Turkish Samsun arose as described by Wildman et al. (1973). Plants were grown under greenhouse conditions for 2-4 months after germination of the seeds, as described by Kawashima and Wildman (1972). Leaves of the polyploid series, based on Burley 21 and Ky 16, and of the male sterile Burley 21 were sent by airmail from Kentucky to Los Angeles. Transporting the leaves by this method does not result in any changes in the patterns obtained on isoelectric focusing. Crystalline fraction 1 protein was obtained from leaves (50 g wet weight) by a remarkably simple procedure (Chan et al., 1972). The leaves of nine plants grown in the same flat from a single seed collection were combined to make up each 50-g batch of leaves. The protein was recrystallized three times before analysis by isoelectric focusing, exactly as described by Kung et al. (1974). The crystalline fraction I proteins were dissolved in 8 Murea and S-carboxymethylated by reaction with iodoacetic acid before application to a prefocused 4.5~ polyacrylamide slab gel containing 1% Ampholine, pH 5-7, and 8 M urea. lsoelectric focusing was performed for 18 hr at 300 V and the polypeptide bands were visualized by staining with bromophenol blue. RESULTS Polypeptide Composition of Fraction I Protein from IV. glauca
The six seed collections of N. glauca, representing the four continents of America, Africa, Asia, and Australia, indicate the widespread distribution of this species in the world. However, as shown in Fig. 1, the polypeptide patterns of the fraction I proteins are similar among the six collections; each protein is made up of three large subunit polypeptides and a single small subunit polypeptide. There have therefore been no gross mutational changes in the fraction [ protein of N. glauca induced by changes in its environment as it spread around the world. Polypeptide Composition of Fraction I Protein from Cuitivars of IV. tabacum
The ten cultivars of N. tabacum analyzed are representative products of the intensive efforts of man to deliberately change the anatomy, cytology, physiology, disease resistance, climatic tolerance, and biochemical quality of the commercial product of a plant that has been under continuous cultivation in most regions of the world for several hundred years. However,
774
Chen, Kung, Gray, and Wildman
Fig. 1. Polypeptide composition of fraction I protein from N. g[auca collections. Polypeptides were separated by isoelectric focusing in polyacrylamide gel slabs containing 8 Murea and 1~ Ampholine,pH 5-7. Fraction I proteins applied at 20/~g per sample. From left to right, N. glauca collected from (1) California; (2) Australia; (3) Israel; (4) Kenya; (5) Mexico; (6) California. Abbreviations: L, large subunit polypeptides; S, small subunit polypeptides.
as shown in Figs. 2 and 3, the polypeptide patterns of the ten cultivars are identical; the protein is composed of three large subunit polypeptides and two small subunit polypeptides. We also analyzed fraction I protein isolated from the white tissue of a variegated mutant of Turkish Samsun and from an isogenic Turkish Samsun, and in each case the polypeptide pattern obtained was identical to the patterns of the other cultivars.
Polypeptide Composition of Fraction I Protein from a Polyploid Series of PC. tabacum
The polyploid series, containing diploid, triploid, and tetraploid plants, was based on the Burley 21 and Ky 16 cultivars. As shown in Fig. 3, there were no differences in the polypeptide patterns between the individual samples and the patterns were similar to those of the other cultivars. This indicates that gene dosage does not affect the polypeptide composition of fraction I protein.
Polypeptides of Fraction I Protein from Nicotiana Cultivars
775
L
Fig. 2. Polypeptide composition of fraction I protein from cultivars of N. tabacum. Polypeptide chains were separated by isoelectric focusing as described in the caption of Fig. 1. Cultivars, from left to right: (1) Turkish Samsun, (2) BY 4, (3) Ky 57, (4) Maryland Mammoth, (5) NC 95, (6) White Daruma, (7) GreenDaruma, (8) Havana 38.
Polypeptide Composition of Fraction I Protein from a Male Sterile N. tabacum
The male sterile line analyzed was a Burley 21 cultivar. The flowers were characterized by the complete absence of anthers but could be fertilized by pollen from normal fertile Burley 21 plants. The analysis of the fraction I protein is shown in Fig. 3, and it can be seen that the polypeptide pattern is different from that of the polyploid series and other eultivars of N. tabacum. The difference is in the position of the large subunit polypeptides, whereas the small subunit polypeptides are identical to those of the other cultivars. The large subunit polypeptides are similar to those of the Australian species of Ni¢otiana (see Sakano et al., 1974). It was deduced from the isoelectric focusing patterns that the male sterile line had been derived from the hybridization of an Australian species as the female parent and N. tabacum as the source of pollen for the fertilization. Repeated backcrossing and introduction of N. tabaeum nuclear genes for the small subunit of fraction I protein would result in the elimination of the Australian-type small subunit polypeptides and the substitution of the two small subunit polypeptides of N. tabacum. The backcrossing, however, would not affect the Australian
776
Chen, Kung, Gray, and Wildman
Fig. 3. Polypeptide composition of fraction I protein from a polyploid series and from a male sterile line of N. tabacum. Polypeptide chains were separated by isoelectric focusing as described in the caption of Fig. 1. Samples, from left to right: (1) diploid (Burley 21), (2) diploid (Ky 16), (3) diploid (F~ hybrid, Burley 21 x Ky 16), (4) triploid (Ky 16 4n x Ky 16 2n), (5) triploid (Burley 21 4n x Ky 16 2n), (6) triploid (Ky 16 4n x Burley 21 2n), (7) tetraploid (Burley 21 4n), (8) male sterile Burley 21. cytoplasm, in particular the chloroplast genes for the large subunit polypeptides, and would give rise to a tobacco line with the nuclear gene characteristics of N. tabacurn. DISCUSSION The present study indicates that in N. gIauca and in normal true breeding N. tabacurn there is no variation in the polypeptide patterns of fraction I protein obtained by isoelectric focusing. Although this finding was not entirely unexpected because the protein was crystallized from a homogenate
Polypeptides of Fraction I Protein from Nicotiana Cultivars
777
of several plants within the species, it was slightly surprising because the seed collections were from plant material that was highly inbred due to selfpollination over many generations and this would be expected to perpetuate any slight variation in the polypeptide pattern. These analyses, therefore, increase our confidence that intraspecies variation will not obscure the results obtained in genetic and evolutionary studies due to interspecies differences. The most surprising result in the present study was the polypeptide composition of the male sterile line of iV. tabacum. However, a search of the breeding record of this particular cultivar (S. J. Sheen, personal communication) revealed that an Australian species, either N. suaveolens or N. megalosiphon, was the female parent in the original cross with N. tabacum, as predicted from the polypeptide pattern. Both Australian species have large subunit polypeptides which correspond to those of the male sterile line and both species have three small subunit polypeptides, none of which corresponds to the two small subunit polypeptides ofN. tabaeum, indicating that the small subunit polypeptides of the male sterile line were indeed derived from the nuclear genome of AT. tabacum. Backcrossing the male sterile cultivar with the two Australian species produced seed only when N. megMosiphon was the pollen source, possibly suggestingthat N, megalosiphon, and not N. suaveolens, was the species involved in the origin of the male sterile line. There are two important consequences of this analysis of fraction I protein from the male sterile Burley 21 cultivar of N. tabacum. First, it demonstrates that in male sterile lines, which are of great advantage to plant breeders because they allow controlled pollination without hand emasculation, the chloroplast genome cannot be altered by conventional breeding techniques. Second, it indicates that in studies on the physiological basis of male sterility greater emphasis ought to be placed on the effects of the chloroplast genome, especially as the mitochondrial genome has been traditionally regarded as the source of the cytoplasmic factor in male sterility.
ACKNOWLEDGMENTS We thank Dr. N. Kawashima of the Hatano Tobacco Experiment Station, Japan Monopoly Corporation, for the gift of seeds, Dr. S. J. Sheen of the Department of Plant Pathology, University of Kentucky, Lexington, for seeds and for shipping the leaves of the polyploid series and the male sterile line of N. tabacum, and Professor C. A. Schroeder of this institution for numerous collections of seeds of N. glauca in various parts of the world. Seeds of the isogenic Turkish Samsun cultivar of N. tabacum were provided by the late J. P. Nitsch.
778
Chen, Kung, Gray, and Wildman REFERENCES
Chan, P. H., and Wildman, S. G. (1972). Chloroplast DNA codes for the primary structure of the large subunit of fraction I protein. Biochim. Biophys. Acta 277:677. Chan, P. H., Sakano, K., Singh, S., and Wildman, S. G. (1972). Crystalline fraction I protein: Preparation in large yield. Science 176:1145. Gray, J. C., Kung, S. D., Wildman, S. G., and Sheen, S. J. (1974). Origin of Nicotiana tabacum L. detected by polypeptide composition of fraction [ protein. Nature 252:226. Kawashima, N. (1969). Comparative studies on fraction I protein from spinach and tobacco leaves. Plant Cell Physiol. 10:31. Kawashima, N., and Wildman, S. G. (1970). Fraction I protein. Ann. Rev. Plant Physiol. 21:325. Kawashima, N., and Wildman, S. G. (1972). Studies on fraction [ protein IV: Mode of inheritance of primary structure in relation to whether chloroplast or nuclear DNA contains the code for a chloroplast protein. Biochim. Biophys. Acta 262:42. Kung, S. D., Sakano, K., and Wildman, S. G. (1974). Multiple peptide composition of the large and small subunits of Nicotiana tabacum fraction I protein ascertained by fingerprinting and electro-focusing. Biochim. Biophys. Acta 365:138. Kung, S. D., Gray, J. C., Wildman S. G., and Carlson, P. S. (1975). Polypeptide composition of fraction I protein from parasexual hybrid plants in the genus Nicotiana. Science 187:353. Nitsch, J. P., and Nitsch, C. (1969). Haploid plants from pollen grains. Science 163:85. Rutner, A. C. (1970). Estimation of the molecular weight of ribulose diphosphate carboxylase sub-units. Biochem. Biophys. Res. Comrnun. 39:923. Rutner, A. C., and Lane, M. D. (1967). Nonidentical subunits of ribulose diphosphate carboxylase. Biochem. Biophys. Res. Commun. 28:531. Sakano, K., Kung, S. D., and Wildman, S. G. (1974). Identification of several chloroplast DNA genes which code for the large subunit of Nicotiana fraction I proteins. Mol. Gen. Genet. 130:91. Wildman, S. G., Lu-Liao, C., and Wong-Staal, F. (1973), Maternal inheritance, cytology and macromolecular composition of defective chloroplasts in a variegated mutant of Nicotiana tabacum. Planta 113:293.
