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The evolution of fraction 1 protein and the distribution of the small subunit polypeptide coding sequences in the genus Brassica
Plant Science Letters, 12 (1978) 299--303 © Elsevier/North-Holland Scientific Publishers Ltd.
299
T H E E V O L U T I O N O F F R A C T I O N 1 P R O T E I N A N D T H E DISTRIBUTION O F T H E S M A L L S U B U N I T P O L Y P E P T I D E C O D I N G S E Q U E N C E S IN T H E GENUS BRASSICA
A.A. G A T E N B Y * and E.C. C O C K I N G Department of Botany, University of Nottingham, University Park, Nottingham N G 7 2 R D (United Kingdom) (Received December 21st, 1977) (Revision received March 10th, 1978) (Accepted March 10th, 1978)
SUMMARY
The sub-unit polypeptide composition of S-carboxymethylated Fraction 1 protein has been examined by isoelectric focusing for Brassica campestris, B. carinata, B. cretica, B. juncea, B. napus, B. nigra, B. oleracea, Raphanobrassica and Raphanus sativus. The small sub-unit polypeptide composition supports the established taxonomic classification in this group and indicates that B. oleracea is an ancient allopolyploid. The molar ratios of the small sub-unit polypeptides has enabled a calculation of the minimum number of nuclear coding sequences for the small sub-unit polypeptides throughout this group of species.
INTRODUCTION
Fraction 1 protein molecules are composed of eight large and eight small sub-units [1] and it has been shown that the small and large sub-units are coded for by nuclear and chloroplast DNA respectively (reviewed in 2). Isoelectric focusing of S-carboxymethylated Fraction 1 protein in 8 M urea resolves the maternally inherited large sub-unit into three polypeptides and the Mendelian inherited small sub-unit into one to four polypeptides, depending on the species [3 ]. In Nicotiana the multiple polypeptide composition of the small sub-units of Fraction 1 protein has been shown to be the consequence of interspecific hybridisation between species with different polypeptides, followed by * Present address: Department of Molecular Biology, University of Edinburgh, Kings Building, Mayfield Road, Edinburgh EH9 3JR (United Kingdom)
300 chromosome doubling to give a fertile amphidiploid species possessing the nuclear coded polypeptides of both parental species [3,4]. The presence of a multiple small sub-unit polypeptide composition therefore suggests an origin by allopolyploidy [5]. MATERIALS AND METHODS The sub-unit polypeptide composition of Fraction 1 protein from the following species was examined: B. campestris (2n = 20, genomic formula aa), four cvs; B. carinata (2n = 34, bbcc); B. cretica (2n = 16, b b ) ; B , oleracea 36, aabb); B. napus (2n = 38, aacc), three cvs; B. nigra (2n = 16,bb); B. oleracea (2n = 18, cc), six cvs; Raphanobrassica (2n = 36, rrcc) and Raphanus sativus (2n = 18, rr). Fraction 1 protein was isolated from young leaves by immunoabsorption as previously described [6] except that an extract of 10 g of leaves was passed through a 2.5 × 12.0 cm column of immobilised antibodies raised against Nicotiana tabacum Fraction I protein. Elution of Fraction 1 protein from the column with 8 M urea, S-carboxymethylation with iodoacetic acid and isoelectric focusing were carried out exactly as described before [6]. In addition Fraction 1 protein from all species was purified by ammonium sulphate fractionation, gel filtration with Sephadex G-25 and G-200, and DEAE-cellulose chromatography [7]. Stained gels were scanned with a JoyceLoebl Mk II Chromoscan recording and integrating densitometer with a transmission optical system at 560--580 nm with a 1 : 9 specimen drive gear ratio and a 4 : 1 Y scale expansion. RESULTS Isoelectric focusing of the S-carboxymethylated Fraction 1 protein with 8 M urea resolved a single small sub-unit polypeptide (polypeptide a) that was shared in c o m m o n by all species. In addition to the shared small sub-unit polypeptide a, B. oleracea possessed a second small subunit polypeptide (polypeptide b) in an equimolar concentration to polypeptide a (Figs. 1, 2 and 3). B. carinata, B. napus and Raphanobrassica also contained the small sub-unit polypeptide b in a proportion of 1 mol of b to 3 mol of a (Figs. 2 and 3). The ratios of the peaks of polypeptides a : b as determined by scanning densitometry were, B. oleracea (53.3 : 46.7),B. carinata {77.0 : 23.0), B. napus {74.4 : 25.6) and Raphanobrassica {74.0 : 26.0). When subjected to a Chi 2 analysis with 1 d.f., and using the integrated values under the peaks, all results were significant at p -- 0.05. Difficulty was encountered in the analysis o f the large subunit polypeptides due to its apparent degradation during purification or S-carboxymethylation, with the generation of additional bands. This problem was n o t overcome by the addition to the homogenisation buffer of 2 mM phenylmethyl sulphonyl fluoride (serine protease inhibitor) and up to 5% (w/v) bovine serum albumin (to prevent polyphenol interactions). However, Uchimiya et al [8] have been able to examine the large sub-unit polypeptide composition in this genus.
301
Fig. 1.
a
a
b
b
Fig.)~3.
Fig. 2.
+
-
+
Fig. 1. The large (L) and small (S) sub-unit polypeptides of S-carboxymethylated Fraction 1 protein from B. oleracea separated by isoelectric focusing with 8 M urea.
Fig. 2. The position of the Fraction 1 protein small sub-unit polypeptides of A; B. oleracea and B; B. napus after electrofocusing with 8 M urea. a refers to polypeptide a and b refers to polypeptide b. Fig. 3. Scanning densitometry of the small sub-unit polypeptides a and b o f Fraction 1 protein after electrofocusing and staining with bromophenol blue. The scan on the left is from B. oleracea and the scan on the right is from B. napus.
DISCUSSION
Cytological research on the various cultivated Brassiea species has provided the basis for a simple t a x o n o m y within the genus in which 3 elementary monogenomic (B. oleracea, B. n~gra, B. campestris) and three amphidiploid digenomic (B. napus, B. carinata, B. juncea) species m a y be distinguished [9]. B. napus arose from a cross between B. oleracea and B. campestris, B. carinata from B. oleracea and B. nigra, and B. juncea from B. carnpestris and B. nigra. These crosses have been made experimentally [10] and a synthetic amphidiploid,
302
Raphanobrassica, has also been obtained by crossing B. oleracea with R. sativus [11 ]. The second small sub-unit polypeptide b in B. oleracea supports the established taxonomic classification in this group [9]. Where B. oleracea had crossed with B. nigra or B. campestris, the resulting amphidiploids, B. carinata and B. napus also contained polypeptide b. B. nigra and B. campestris contain only the small sub-unit polypeptide a and the resultant amphidiploid between them, B. ]uncea, also contains only polypeptide a. Uchimiya et al. [8] by examining the large sub-unit polypeptides have demonstrated that B. ]uncea arose from a cross between B. campestris 9 and B. nigra d, and that B. carinata arose from B. nigra ? and B. oleracea d. The large sub-unit polypeptides of B. campestris and B. oleracea had similar isoelectric points, thus preventing the direction of the cross giving rise to B. napus to be determined. The two small sub-unit polypeptides a and b in B. oleracea suggest that it is an ancient allopolyploid. This is supported by cytological observations where studies on secondary association of chromosomes have been interpreted as showing a basic chromosome number for the genus Brassica of x = 5 (12). This implies that B. oleracea is an allopolyploid from a cross between 2 primitive 5-chromosome species with the subsequent loss of a pair of chromosomes [13]. The suggested allopolyploid origin of B. oleracea may account for the polymorphic characteristics of this species when compared to other Brassica species. The equimolar concentrations of polypeptides a and b in B. oleracea implies that the 2 primitive species giving rise to it each contributed equal coding sequences for their small sub-unit polypeptides. Given that the basic chromosome number in the genus is x = 5 [12], then the basic number must contain a minimum of one coding sequence for a small subunit polypeptide. B. campestris with 2n = 20 has 4 complements of the basic chromosome number and should contain a minimum of 4 coding sequences for polypeptide a. Similarly B. oleracea would be expected to have a minimum of 4 coding sequences, but 2 would code for polypeptide a and 2 for polypeptide b to give the equimolar' ratio of polypeptides observed in this species. In the amphidiploid B. napus derived from B. oleracea × B. campestris, the minimum number of coding sequences would be 8, 6 of which would code for polypeptide a and 2 for polypeptide b. This is supported by the 3 : 1 molar ratio of small sub-unit polypeptides a : b observed in B. napus. A similar explanation would account for the 3 : 1 molar ratios of small sub-unit polypeptides a : b found in the amphidiploids B. carinata and Raphanobrassica, on the assumption that all coding sequences are equally transcribed and translated. The number of coding sequences for the small sub-unit polypeptides increases during auto- or allopolyploidy, but the a m o u n t of Fraction 1 protein in leaves does not increase on a fresh wt.basis, as observed by purification yield. It would appear that the increase in coding capacity by polyploidy results in reduced utilisation of those parts of the genome coding for the small sub-unit polypeptides, and that this reduction is spread equally among the coding sequences. If the ratio of coding sequences for polypeptides a and b can be taken as a
303
measure of the entire nuclear genome, then support for the coding capacity data might be expected from the breeding behaviour of the amphidiploid species. The chromosomes of B. campestris are 4 basic copies possibly arrived at by autopolyploidy whereas the chromosomes of B. oleracea are possibly from two primitive species, one of which may have had a c o m m o n origin to B. campestris as shown by the small sub-unit polypeptide composition. The nuclear genome of the amphidiploid B. napus would therefore be predisposed in favour of B. campestris and it might be anticipated that B. napus would cross more readily with B. campestris than with B. oleracea. Data from Yarnell [10] shows that the crossing of B. napus with B. campestris is easier to achieve than between B. napus and B. oleracea. Similarly the nuclear genomes of B. carinata and Raphanobrassica would be predisposed towards B. nigra and R. sativus respectively. It has been found that more hybrids are obtained between B. carinata and B. nigra than with B. oleracea, and Raphanobrassica forms seed more readily with R. sativus than with B. oleracea (10). The observed breeding behaviour of these species is thus in agreement with the predicted breeding behaviour as expected from the small sub-unit polypeptide composition of Fraction 1 protein. ACKNOWLEDGEMENTS
We are grateful for the generous gifts of seeds from J.S. Hemingway, Colman Foods; The National Institute of Agricultural Botany; I.H. McNaughton, Scottish Plant Breeding Station; The Royal Botanic Gardens, Kew; Suttons Seeds Ltd., and the Swedish Seed Association. REFERENCES
1 2 3 4 5 6 7 8 9 10 11 12
T.S. Baker, D. Eisenberg and F. Eiserling, Science, 196 (1977) 293. S.D. Kung, Ann. Rev. Plant Physiol., 28 (1977) 401. K. Sakano, S.D. Kung and S.G. Wildman, Mol. Gen. Genet., 130 (1974) 91. S.D. Kung, K. Sakano, J.C. Gray and S.G. Wildman, J. Mol. Evol., 7 (1975) 59. K. Chen, S.D. Kung, J.C. Gray and S.G. Wildman, Plant Sci. Lett., 7 (1976) 429. A.A. Gatenby and E.C. Cocking, Plant Sci. Lett., 10 (1977) 97. A.A. Gatenby and E.C. Cocking, Plant Sci. Lett., 12 (1978) 177. H. Uchimiya, K. Chen and S.G. Wildman, Stadler Symp., 9 (1978) In press. N.U. Jap, J. Bot., 7 (1935) 389. S.H. Yarnell, Bot. Rev., 22 (1956) 81. I.H. McNaughton, Euphytica, 22 (1973) 70. K.F. Thompson, Cabbages, Kales etc., in N.W. Simmonds (Ed.), Evolution of Crop Plants, Longman, London, 1976, p. 49. 13 S.M. Sikka, J. Genet., 40 (1940) 441.
299
T H E E V O L U T I O N O F F R A C T I O N 1 P R O T E I N A N D T H E DISTRIBUTION O F T H E S M A L L S U B U N I T P O L Y P E P T I D E C O D I N G S E Q U E N C E S IN T H E GENUS BRASSICA
A.A. G A T E N B Y * and E.C. C O C K I N G Department of Botany, University of Nottingham, University Park, Nottingham N G 7 2 R D (United Kingdom) (Received December 21st, 1977) (Revision received March 10th, 1978) (Accepted March 10th, 1978)
SUMMARY
The sub-unit polypeptide composition of S-carboxymethylated Fraction 1 protein has been examined by isoelectric focusing for Brassica campestris, B. carinata, B. cretica, B. juncea, B. napus, B. nigra, B. oleracea, Raphanobrassica and Raphanus sativus. The small sub-unit polypeptide composition supports the established taxonomic classification in this group and indicates that B. oleracea is an ancient allopolyploid. The molar ratios of the small sub-unit polypeptides has enabled a calculation of the minimum number of nuclear coding sequences for the small sub-unit polypeptides throughout this group of species.
INTRODUCTION
Fraction 1 protein molecules are composed of eight large and eight small sub-units [1] and it has been shown that the small and large sub-units are coded for by nuclear and chloroplast DNA respectively (reviewed in 2). Isoelectric focusing of S-carboxymethylated Fraction 1 protein in 8 M urea resolves the maternally inherited large sub-unit into three polypeptides and the Mendelian inherited small sub-unit into one to four polypeptides, depending on the species [3 ]. In Nicotiana the multiple polypeptide composition of the small sub-units of Fraction 1 protein has been shown to be the consequence of interspecific hybridisation between species with different polypeptides, followed by * Present address: Department of Molecular Biology, University of Edinburgh, Kings Building, Mayfield Road, Edinburgh EH9 3JR (United Kingdom)
300 chromosome doubling to give a fertile amphidiploid species possessing the nuclear coded polypeptides of both parental species [3,4]. The presence of a multiple small sub-unit polypeptide composition therefore suggests an origin by allopolyploidy [5]. MATERIALS AND METHODS The sub-unit polypeptide composition of Fraction 1 protein from the following species was examined: B. campestris (2n = 20, genomic formula aa), four cvs; B. carinata (2n = 34, bbcc); B. cretica (2n = 16, b b ) ; B , oleracea 36, aabb); B. napus (2n = 38, aacc), three cvs; B. nigra (2n = 16,bb); B. oleracea (2n = 18, cc), six cvs; Raphanobrassica (2n = 36, rrcc) and Raphanus sativus (2n = 18, rr). Fraction 1 protein was isolated from young leaves by immunoabsorption as previously described [6] except that an extract of 10 g of leaves was passed through a 2.5 × 12.0 cm column of immobilised antibodies raised against Nicotiana tabacum Fraction I protein. Elution of Fraction 1 protein from the column with 8 M urea, S-carboxymethylation with iodoacetic acid and isoelectric focusing were carried out exactly as described before [6]. In addition Fraction 1 protein from all species was purified by ammonium sulphate fractionation, gel filtration with Sephadex G-25 and G-200, and DEAE-cellulose chromatography [7]. Stained gels were scanned with a JoyceLoebl Mk II Chromoscan recording and integrating densitometer with a transmission optical system at 560--580 nm with a 1 : 9 specimen drive gear ratio and a 4 : 1 Y scale expansion. RESULTS Isoelectric focusing of the S-carboxymethylated Fraction 1 protein with 8 M urea resolved a single small sub-unit polypeptide (polypeptide a) that was shared in c o m m o n by all species. In addition to the shared small sub-unit polypeptide a, B. oleracea possessed a second small subunit polypeptide (polypeptide b) in an equimolar concentration to polypeptide a (Figs. 1, 2 and 3). B. carinata, B. napus and Raphanobrassica also contained the small sub-unit polypeptide b in a proportion of 1 mol of b to 3 mol of a (Figs. 2 and 3). The ratios of the peaks of polypeptides a : b as determined by scanning densitometry were, B. oleracea (53.3 : 46.7),B. carinata {77.0 : 23.0), B. napus {74.4 : 25.6) and Raphanobrassica {74.0 : 26.0). When subjected to a Chi 2 analysis with 1 d.f., and using the integrated values under the peaks, all results were significant at p -- 0.05. Difficulty was encountered in the analysis o f the large subunit polypeptides due to its apparent degradation during purification or S-carboxymethylation, with the generation of additional bands. This problem was n o t overcome by the addition to the homogenisation buffer of 2 mM phenylmethyl sulphonyl fluoride (serine protease inhibitor) and up to 5% (w/v) bovine serum albumin (to prevent polyphenol interactions). However, Uchimiya et al [8] have been able to examine the large sub-unit polypeptide composition in this genus.
301
Fig. 1.
a
a
b
b
Fig.)~3.
Fig. 2.
+
-
+
Fig. 1. The large (L) and small (S) sub-unit polypeptides of S-carboxymethylated Fraction 1 protein from B. oleracea separated by isoelectric focusing with 8 M urea.
Fig. 2. The position of the Fraction 1 protein small sub-unit polypeptides of A; B. oleracea and B; B. napus after electrofocusing with 8 M urea. a refers to polypeptide a and b refers to polypeptide b. Fig. 3. Scanning densitometry of the small sub-unit polypeptides a and b o f Fraction 1 protein after electrofocusing and staining with bromophenol blue. The scan on the left is from B. oleracea and the scan on the right is from B. napus.
DISCUSSION
Cytological research on the various cultivated Brassiea species has provided the basis for a simple t a x o n o m y within the genus in which 3 elementary monogenomic (B. oleracea, B. n~gra, B. campestris) and three amphidiploid digenomic (B. napus, B. carinata, B. juncea) species m a y be distinguished [9]. B. napus arose from a cross between B. oleracea and B. campestris, B. carinata from B. oleracea and B. nigra, and B. juncea from B. carnpestris and B. nigra. These crosses have been made experimentally [10] and a synthetic amphidiploid,
302
Raphanobrassica, has also been obtained by crossing B. oleracea with R. sativus [11 ]. The second small sub-unit polypeptide b in B. oleracea supports the established taxonomic classification in this group [9]. Where B. oleracea had crossed with B. nigra or B. campestris, the resulting amphidiploids, B. carinata and B. napus also contained polypeptide b. B. nigra and B. campestris contain only the small sub-unit polypeptide a and the resultant amphidiploid between them, B. ]uncea, also contains only polypeptide a. Uchimiya et al. [8] by examining the large sub-unit polypeptides have demonstrated that B. ]uncea arose from a cross between B. campestris 9 and B. nigra d, and that B. carinata arose from B. nigra ? and B. oleracea d. The large sub-unit polypeptides of B. campestris and B. oleracea had similar isoelectric points, thus preventing the direction of the cross giving rise to B. napus to be determined. The two small sub-unit polypeptides a and b in B. oleracea suggest that it is an ancient allopolyploid. This is supported by cytological observations where studies on secondary association of chromosomes have been interpreted as showing a basic chromosome number for the genus Brassica of x = 5 (12). This implies that B. oleracea is an allopolyploid from a cross between 2 primitive 5-chromosome species with the subsequent loss of a pair of chromosomes [13]. The suggested allopolyploid origin of B. oleracea may account for the polymorphic characteristics of this species when compared to other Brassica species. The equimolar concentrations of polypeptides a and b in B. oleracea implies that the 2 primitive species giving rise to it each contributed equal coding sequences for their small sub-unit polypeptides. Given that the basic chromosome number in the genus is x = 5 [12], then the basic number must contain a minimum of one coding sequence for a small subunit polypeptide. B. campestris with 2n = 20 has 4 complements of the basic chromosome number and should contain a minimum of 4 coding sequences for polypeptide a. Similarly B. oleracea would be expected to have a minimum of 4 coding sequences, but 2 would code for polypeptide a and 2 for polypeptide b to give the equimolar' ratio of polypeptides observed in this species. In the amphidiploid B. napus derived from B. oleracea × B. campestris, the minimum number of coding sequences would be 8, 6 of which would code for polypeptide a and 2 for polypeptide b. This is supported by the 3 : 1 molar ratio of small sub-unit polypeptides a : b observed in B. napus. A similar explanation would account for the 3 : 1 molar ratios of small sub-unit polypeptides a : b found in the amphidiploids B. carinata and Raphanobrassica, on the assumption that all coding sequences are equally transcribed and translated. The number of coding sequences for the small sub-unit polypeptides increases during auto- or allopolyploidy, but the a m o u n t of Fraction 1 protein in leaves does not increase on a fresh wt.basis, as observed by purification yield. It would appear that the increase in coding capacity by polyploidy results in reduced utilisation of those parts of the genome coding for the small sub-unit polypeptides, and that this reduction is spread equally among the coding sequences. If the ratio of coding sequences for polypeptides a and b can be taken as a
303
measure of the entire nuclear genome, then support for the coding capacity data might be expected from the breeding behaviour of the amphidiploid species. The chromosomes of B. campestris are 4 basic copies possibly arrived at by autopolyploidy whereas the chromosomes of B. oleracea are possibly from two primitive species, one of which may have had a c o m m o n origin to B. campestris as shown by the small sub-unit polypeptide composition. The nuclear genome of the amphidiploid B. napus would therefore be predisposed in favour of B. campestris and it might be anticipated that B. napus would cross more readily with B. campestris than with B. oleracea. Data from Yarnell [10] shows that the crossing of B. napus with B. campestris is easier to achieve than between B. napus and B. oleracea. Similarly the nuclear genomes of B. carinata and Raphanobrassica would be predisposed towards B. nigra and R. sativus respectively. It has been found that more hybrids are obtained between B. carinata and B. nigra than with B. oleracea, and Raphanobrassica forms seed more readily with R. sativus than with B. oleracea (10). The observed breeding behaviour of these species is thus in agreement with the predicted breeding behaviour as expected from the small sub-unit polypeptide composition of Fraction 1 protein. ACKNOWLEDGEMENTS
We are grateful for the generous gifts of seeds from J.S. Hemingway, Colman Foods; The National Institute of Agricultural Botany; I.H. McNaughton, Scottish Plant Breeding Station; The Royal Botanic Gardens, Kew; Suttons Seeds Ltd., and the Swedish Seed Association. REFERENCES
1 2 3 4 5 6 7 8 9 10 11 12
T.S. Baker, D. Eisenberg and F. Eiserling, Science, 196 (1977) 293. S.D. Kung, Ann. Rev. Plant Physiol., 28 (1977) 401. K. Sakano, S.D. Kung and S.G. Wildman, Mol. Gen. Genet., 130 (1974) 91. S.D. Kung, K. Sakano, J.C. Gray and S.G. Wildman, J. Mol. Evol., 7 (1975) 59. K. Chen, S.D. Kung, J.C. Gray and S.G. Wildman, Plant Sci. Lett., 7 (1976) 429. A.A. Gatenby and E.C. Cocking, Plant Sci. Lett., 10 (1977) 97. A.A. Gatenby and E.C. Cocking, Plant Sci. Lett., 12 (1978) 177. H. Uchimiya, K. Chen and S.G. Wildman, Stadler Symp., 9 (1978) In press. N.U. Jap, J. Bot., 7 (1935) 389. S.H. Yarnell, Bot. Rev., 22 (1956) 81. I.H. McNaughton, Euphytica, 22 (1973) 70. K.F. Thompson, Cabbages, Kales etc., in N.W. Simmonds (Ed.), Evolution of Crop Plants, Longman, London, 1976, p. 49. 13 S.M. Sikka, J. Genet., 40 (1940) 441.