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Isoelectrofocusing of non-carboxymethylated fraction I protein from green callus
Plant Science Letters, 25 (1982) 37--41
37
Elsevier/North-Holland Scientific Publishers Ltd.
ISOELECTROFOCUSING OF NON-CARBOXYMETHYLATED FRACTION I PROTEIN FROM GREEN CALLUS
A. HIRAI
Graduate Division of Biochemical Regulation, Faculty of Agriculture, Nagoya University, Nagoya, 464 (Japan)
(Received April 10th, 1981) (Revision received June 1st, 1981 )
(Accepted October 5th, 1981)
SUMMARY
Fraction I protein, a useful genetic marker for nuclear and chloroplast genomes, has been examined by isoelectrofocusing, with or without carboxymethylation. The result that both methods express relatively the same pattern shows that carboxymethylation of this protein is not necessary. The technique developed, without carboxymethylation, permitted analysis of fraction I protein from green callus.
INTRODUCTION
Fraction I protein is a major soluble protein in green leaves [1] and has enzymatic activities of ribulose-l,5-bisphosphate carboxylase and oxygenase [2]. It consists of two subunits, namely large and small subunits, coded by chloroplast [3] and nuclear [4] DNA, respectively. The polypeptide composition of S-carboxymethylated fraction I protein, revealed by isoelectrofocusing in 8 M urea, shows a unique composition for each plant species in many cases, serving as a genetic marker for chloroplast and nuclear genomes [5--7]. Analysis by this method has been widely used in Nicotiana and other genera. However, according to Kung et al. [ 5 ], carboxymethylation of the protein is necessary for isoelectrofocusing; therefore the procedure for analysis is rather complicated. Uchimiya et al. developed the micro method by using antibody against fraction I protein [8]. Although this simplifies the procedure greatly, it is still difficult to handle the small amount of fraction I protein. The purpose of this letter is to show that carboxymethylation of the protein is not necessary for analysis by electrofocusing, and to show that this simpler m e t h o d can be adapted to small amounts of leaf tissue also to 0304--4211/82/0000--0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publ.ishers Ltd.
38 small pieces of green callus, which contain only a very low quantity of fraction I protein. MATERIALS AND METHODS
Plant materials Plants were grown in a green house. Soybean (Glycine max L.) callus was initiated from aseptically grown hypocotyl in Murashige-Skoog medium [9] supplemented with 2 mg/l 2, 4-D and 0.2 rag/1 kinetin. The white callus was then transferred to the same medium with different plant hormones, 4 mg/l NAA and 2.5 mg/1 kinetin. The green callus was obtained in 3--4 weeks after transplant. Tobacco (Nicotiana tabacurn L.) green callus was also obtained by the same procedure except that white callus was initiated from sterilized seed. Isoelectrofocusing of fraction I protein The method of Uchimiya et al. [8] was used with modifications. A quantity of leaf (0.1 g) or 0.5 g of green callus tissue, was ground with mortar and pestle in 1 ml of buffer B (Ref. 10, 25 mM Tris--HC1 pH 7.4, 200 mM NaC1, 0.5 mM EDTA) containing 100 mM ~-mercaptoethanol and 0.5% sodium ascorbate. The homogenate was centrifuged at 10 000 × g for 4 rain and supernatant was passed through a Sephadex G.25 column equilibrated with buffer B. This procedure could be omitted in the case of young leaves, but it is necessary in callus or old leaves. Void volume fraction or ground homogenate was centrifuged at 20 000 × g for 20 min and the supernatant was incubated at 37°C for 2 h and at 4°C overnight with antiserum prepared against tobacco crystalline fraction I protein. The antibodyfraction I protein complex was precipitated by centrifugation and washed twice with buffer B. The complex, in a test tube, was dissolved in 30--70 ul of buffer containing 0.5 M Tris--HCl (pH 8.5), 1 mM EDTA, 8 M urea and 50 mg/ml of dithiothreitol and sealed with a serum cap. The test tube was evacuated and then filled with N: gas using a hypodermic needle through a serum cap, and kept at 25°C for 2 h. The protein solution was then passed through Sephadex G-25 equilibrated with 8 M urea in 0.5 M Tris--HCl (pH 8.5), by the centrifugation method [11]. The protein fraction at the bottom of the centrifugation tube was immediately isoelectrofocused using the technique of Kung et al. [5]. When S-carboxymethylation was carried out, 50 ~1 of 8 M urea in 0.5 M Tris--HCl (pH 8.5) containing 7 mg of iodoacetic acid was injected into the aluminium foil covered test tube at 10 rain, before gel filtration. RESULTS AND DISCUSSION
Analysis without carboxymethylation To examine the necessity of S-carboxymethylation of fraction I protein for isoelectrofocusing, the proteins from leaves of three Nicotiana species were
39
<-----A 1 2
> ,5
< 1
B 2
> ,5
A 1
B 1
Fig. 1. Isoelectrofocusing resolution of non-carboxymethylated (A) and carboxymethylated (B) fraction I protein from N. tabacum (1), N. glutinosa (2) and N. glauca (3).
analyzed by electrofocusing, with or without carboxymethylation. The results in Fig. 1 show that without carboxymethylation, all bands stayed at the higher p H region compared to the carboxymethylated fraction I proteins. The large subunits also resolved into three bands, with or without carboxymethy]ation. Although it is not known w h y they resolved into three bands [12 ], it is clear that this is not because of artifactscaused by carboxymethylation. The pattern, without carboxymethylation, did not depend on the kind of carrierampholyte (e.g.Pharmalyte was used in Fig. 1 and Ampholine in Fig. 2). Crystallinefraction I protein was also analyzed without carboxy-
40
® <--- A
1
-~
2
~"-B
1
=
2
®
Fig. 2. Comparison of electrofoemfing pattern of non-earboxymethylated fraction I proteins from leaf and green callus. (a) Leaf (A) and callus (B) fraction I protein from N. tabacum. (1) Without gel filtration prior to electrofoeudng; (2) With gel filtration prior to electrofocusing. (b) Leaf (A) and callus (B) fraction I protein from soybean.
methylation, and the result was the same. Cammaerts and Jacobs purified fraction I protein by gel electrophoresis and applied it to isoelectrofocusing without carboxymethylation [13]. My results agree with their results and both show that the protection of fraction I protein by S-carboxymethylation is not necessary for obtaining reproducible patterns by isoelectrofocusing. Another advantage of this m e t h o d is its simplicity.
Electro focusing of fraction I protein from green callus Without carboxymethylation, the procedure for electrofocusing becomes simpler and the a m o u n t of tissue required for analysis is smaller. Therefore,
41 I tried to analyzed fraction I proteins from green callus, because it is important to develop the technique by which genetic marker can be analyzed at the stage of callus tissue. The concentration of the protein in green callus is much lower than that in leaves. Morever, it also contains more phenolic compounds which disturb the analysis. This difficulty was overcome by the m e t h o d described in Materials and Methods. The results were shown in Fig. 2. Fraction I proteins from tobacco and soybean calli show that same pattern as those from leaves. This result strongly supports the view of Gatenby et al. [14] who stated that fraction I protein electrofocusing pattern is a more stable marker than isoenzymes. To analyzed fraction I protein at the stage of callus has m a n y advantages. It is possible to analyze the genetic marker in tissue culture cells, such as soybean which cannot as y e t be regenerated into plantlets (Fig. 2b). Even in the case of callus which can be regenerated, it is often better to analyze the marker in callus tissue, because it saves time. ACKNOWLEDGEMENTS The author thanks Professor S.G. Wildman (University of California, Los Angeles, U.S.A.) for encouragement in fraction I protein research and Dr. H. Uchimiya, Tsukuba University, Japan, for teaching him isoelectrofocusing techniques. This research was partly supported by a Grant-in-Aid for Scientific Research D-566035 from The Ministry of Education, Science and Culture, and the Ishida F o u n d a t i o n (55-205}. REFERENCES
1 2 3 4 5 6 7 8 9 10 11 12 13 14
S.G. Wildman, Arch. Biochem. Biophys., 196 (1979) 598. T.J. Andrews, G.H. Lorimer and N.E. Tolbert, Biochemistry, 12 (1973) 11. P.H. Chan and S.G. Wildman, Biochim. Biophys. Acta, 277 (1972) 677. N. Kawashima and S.G. Wildman, Biochim. Biophys. Acta, 262 (1972) 42. S.D. Kung, K. Sakano and S.G. Wildman, Biochim. Biophys. Acta, 365 (1974) 138. K. Sakano, S.D. Kung and S.G. Wildman, Mol. Gen. Genet., 130 (1974) 91. S.D. Kung, Science, 191 (1976) 429. H. Uchimiya, K. Chen and S.G. Wildman, Plant Sci. Lett., 14 (1979) 387. T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473. P.H. Chan, K. Sakano, S. Singh and S.G. Wildman, Science, 176 (1972) 1145. M.W. Neal and J.R. Florini, Anal. Biochem., 55 (1973) 328. J.C. Gray, S.D. Kung and S.G. Wildman, Arch. Biochem. Biophys., 185 (1978) 272. D. Cammaerts and M. Jacobs, Anal. Biochem., 109 (1980) 317. A.A. Gatenby, F.J. Zapata and E.C. Cocking, Z. Pflanzenzuchtg., 84 (1980) 1.
37
Elsevier/North-Holland Scientific Publishers Ltd.
ISOELECTROFOCUSING OF NON-CARBOXYMETHYLATED FRACTION I PROTEIN FROM GREEN CALLUS
A. HIRAI
Graduate Division of Biochemical Regulation, Faculty of Agriculture, Nagoya University, Nagoya, 464 (Japan)
(Received April 10th, 1981) (Revision received June 1st, 1981 )
(Accepted October 5th, 1981)
SUMMARY
Fraction I protein, a useful genetic marker for nuclear and chloroplast genomes, has been examined by isoelectrofocusing, with or without carboxymethylation. The result that both methods express relatively the same pattern shows that carboxymethylation of this protein is not necessary. The technique developed, without carboxymethylation, permitted analysis of fraction I protein from green callus.
INTRODUCTION
Fraction I protein is a major soluble protein in green leaves [1] and has enzymatic activities of ribulose-l,5-bisphosphate carboxylase and oxygenase [2]. It consists of two subunits, namely large and small subunits, coded by chloroplast [3] and nuclear [4] DNA, respectively. The polypeptide composition of S-carboxymethylated fraction I protein, revealed by isoelectrofocusing in 8 M urea, shows a unique composition for each plant species in many cases, serving as a genetic marker for chloroplast and nuclear genomes [5--7]. Analysis by this method has been widely used in Nicotiana and other genera. However, according to Kung et al. [ 5 ], carboxymethylation of the protein is necessary for isoelectrofocusing; therefore the procedure for analysis is rather complicated. Uchimiya et al. developed the micro method by using antibody against fraction I protein [8]. Although this simplifies the procedure greatly, it is still difficult to handle the small amount of fraction I protein. The purpose of this letter is to show that carboxymethylation of the protein is not necessary for analysis by electrofocusing, and to show that this simpler m e t h o d can be adapted to small amounts of leaf tissue also to 0304--4211/82/0000--0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publ.ishers Ltd.
38 small pieces of green callus, which contain only a very low quantity of fraction I protein. MATERIALS AND METHODS
Plant materials Plants were grown in a green house. Soybean (Glycine max L.) callus was initiated from aseptically grown hypocotyl in Murashige-Skoog medium [9] supplemented with 2 mg/l 2, 4-D and 0.2 rag/1 kinetin. The white callus was then transferred to the same medium with different plant hormones, 4 mg/l NAA and 2.5 mg/1 kinetin. The green callus was obtained in 3--4 weeks after transplant. Tobacco (Nicotiana tabacurn L.) green callus was also obtained by the same procedure except that white callus was initiated from sterilized seed. Isoelectrofocusing of fraction I protein The method of Uchimiya et al. [8] was used with modifications. A quantity of leaf (0.1 g) or 0.5 g of green callus tissue, was ground with mortar and pestle in 1 ml of buffer B (Ref. 10, 25 mM Tris--HC1 pH 7.4, 200 mM NaC1, 0.5 mM EDTA) containing 100 mM ~-mercaptoethanol and 0.5% sodium ascorbate. The homogenate was centrifuged at 10 000 × g for 4 rain and supernatant was passed through a Sephadex G.25 column equilibrated with buffer B. This procedure could be omitted in the case of young leaves, but it is necessary in callus or old leaves. Void volume fraction or ground homogenate was centrifuged at 20 000 × g for 20 min and the supernatant was incubated at 37°C for 2 h and at 4°C overnight with antiserum prepared against tobacco crystalline fraction I protein. The antibodyfraction I protein complex was precipitated by centrifugation and washed twice with buffer B. The complex, in a test tube, was dissolved in 30--70 ul of buffer containing 0.5 M Tris--HCl (pH 8.5), 1 mM EDTA, 8 M urea and 50 mg/ml of dithiothreitol and sealed with a serum cap. The test tube was evacuated and then filled with N: gas using a hypodermic needle through a serum cap, and kept at 25°C for 2 h. The protein solution was then passed through Sephadex G-25 equilibrated with 8 M urea in 0.5 M Tris--HCl (pH 8.5), by the centrifugation method [11]. The protein fraction at the bottom of the centrifugation tube was immediately isoelectrofocused using the technique of Kung et al. [5]. When S-carboxymethylation was carried out, 50 ~1 of 8 M urea in 0.5 M Tris--HCl (pH 8.5) containing 7 mg of iodoacetic acid was injected into the aluminium foil covered test tube at 10 rain, before gel filtration. RESULTS AND DISCUSSION
Analysis without carboxymethylation To examine the necessity of S-carboxymethylation of fraction I protein for isoelectrofocusing, the proteins from leaves of three Nicotiana species were
39
<-----A 1 2
> ,5
< 1
B 2
> ,5
A 1
B 1
Fig. 1. Isoelectrofocusing resolution of non-carboxymethylated (A) and carboxymethylated (B) fraction I protein from N. tabacum (1), N. glutinosa (2) and N. glauca (3).
analyzed by electrofocusing, with or without carboxymethylation. The results in Fig. 1 show that without carboxymethylation, all bands stayed at the higher p H region compared to the carboxymethylated fraction I proteins. The large subunits also resolved into three bands, with or without carboxymethy]ation. Although it is not known w h y they resolved into three bands [12 ], it is clear that this is not because of artifactscaused by carboxymethylation. The pattern, without carboxymethylation, did not depend on the kind of carrierampholyte (e.g.Pharmalyte was used in Fig. 1 and Ampholine in Fig. 2). Crystallinefraction I protein was also analyzed without carboxy-
40
® <--- A
1
-~
2
~"-B
1
=
2
®
Fig. 2. Comparison of electrofoemfing pattern of non-earboxymethylated fraction I proteins from leaf and green callus. (a) Leaf (A) and callus (B) fraction I protein from N. tabacum. (1) Without gel filtration prior to electrofoeudng; (2) With gel filtration prior to electrofocusing. (b) Leaf (A) and callus (B) fraction I protein from soybean.
methylation, and the result was the same. Cammaerts and Jacobs purified fraction I protein by gel electrophoresis and applied it to isoelectrofocusing without carboxymethylation [13]. My results agree with their results and both show that the protection of fraction I protein by S-carboxymethylation is not necessary for obtaining reproducible patterns by isoelectrofocusing. Another advantage of this m e t h o d is its simplicity.
Electro focusing of fraction I protein from green callus Without carboxymethylation, the procedure for electrofocusing becomes simpler and the a m o u n t of tissue required for analysis is smaller. Therefore,
41 I tried to analyzed fraction I proteins from green callus, because it is important to develop the technique by which genetic marker can be analyzed at the stage of callus tissue. The concentration of the protein in green callus is much lower than that in leaves. Morever, it also contains more phenolic compounds which disturb the analysis. This difficulty was overcome by the m e t h o d described in Materials and Methods. The results were shown in Fig. 2. Fraction I proteins from tobacco and soybean calli show that same pattern as those from leaves. This result strongly supports the view of Gatenby et al. [14] who stated that fraction I protein electrofocusing pattern is a more stable marker than isoenzymes. To analyzed fraction I protein at the stage of callus has m a n y advantages. It is possible to analyze the genetic marker in tissue culture cells, such as soybean which cannot as y e t be regenerated into plantlets (Fig. 2b). Even in the case of callus which can be regenerated, it is often better to analyze the marker in callus tissue, because it saves time. ACKNOWLEDGEMENTS The author thanks Professor S.G. Wildman (University of California, Los Angeles, U.S.A.) for encouragement in fraction I protein research and Dr. H. Uchimiya, Tsukuba University, Japan, for teaching him isoelectrofocusing techniques. This research was partly supported by a Grant-in-Aid for Scientific Research D-566035 from The Ministry of Education, Science and Culture, and the Ishida F o u n d a t i o n (55-205}. REFERENCES
1 2 3 4 5 6 7 8 9 10 11 12 13 14
S.G. Wildman, Arch. Biochem. Biophys., 196 (1979) 598. T.J. Andrews, G.H. Lorimer and N.E. Tolbert, Biochemistry, 12 (1973) 11. P.H. Chan and S.G. Wildman, Biochim. Biophys. Acta, 277 (1972) 677. N. Kawashima and S.G. Wildman, Biochim. Biophys. Acta, 262 (1972) 42. S.D. Kung, K. Sakano and S.G. Wildman, Biochim. Biophys. Acta, 365 (1974) 138. K. Sakano, S.D. Kung and S.G. Wildman, Mol. Gen. Genet., 130 (1974) 91. S.D. Kung, Science, 191 (1976) 429. H. Uchimiya, K. Chen and S.G. Wildman, Plant Sci. Lett., 14 (1979) 387. T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473. P.H. Chan, K. Sakano, S. Singh and S.G. Wildman, Science, 176 (1972) 1145. M.W. Neal and J.R. Florini, Anal. Biochem., 55 (1973) 328. J.C. Gray, S.D. Kung and S.G. Wildman, Arch. Biochem. Biophys., 185 (1978) 272. D. Cammaerts and M. Jacobs, Anal. Biochem., 109 (1980) 317. A.A. Gatenby, F.J. Zapata and E.C. Cocking, Z. Pflanzenzuchtg., 84 (1980) 1.