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oxygenase in maize and spinach
Plant Science, 74 (1991)1--6 Elsevier Scientific Publishers Ireland Ltd.
1
Multiple forms of the small subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase in maize and spinach Lifen Ren, Johann Salnikow and Joachim Vater Technische Universitdt Berlin, Institiit fiir Biochemie und Molekulare Biologic, Franklinstr. 29, D-IO00 Berlin 10 (Germany) (Received June 1st, 1990; revision received October 22nd, 1990; accepted October 22nd, 1990)
Native small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) from maize has been electrophoretically resolved into five isoforms of identical molecular weight possessing, however, different isoelectric points (pl = 7.4, 7.2, 6.5, 5.7 and 5.2). Gene expression studies (Sheen and Bogorad, EMBO J., 5 (1986) 3417) and proteinchemical sequencing (Ren et al., Biol. Chem. HoppeSeyler, 369 (1988) 609) suggest that the observed isoforms have slightly different amino acid sequences as a result of a multigene expression. For spinach rubisco the native small subunit resolves into two isoforms with p l = 6.6 and 7.2.
Key words: ribulose-l,5-bisphosphate; carboxylase/oxygenase; isoforms
Introduction
Ribulose-l,5-bisphosphate carboxylase/oxygenase ('rubisco') as the central enzyme of photosynthetic carbon dioxide fixation is present in all plants and photosynthetic microorganisms. Its architecture in plants is formed by the assembly of eight large subunits carrying the catalytic active residues and eight small subunits. Whereas the large subunits are plastome coded, the genes for the small subunits (rbcS) are located in the nucleus and translated into precursor polypeptides carrying an amino-terminal extension which serves as the recognition signal for the chloroplast organelle. One of the earliest examples for small subunit multiplicity concerns the tomato plant; Kung et al. [1] demonstrated in 1974 by isoelectric focusing and fingerprinting two discernible polypeptide chains for the small subunit from Nicotiana tabacum, M/iller et al. [2] confirmed by protein chemical sequencing the existence of two slightly different polypeptide Correspondence to: Johann Salnikow, Technische Universit/it Berlin, Instittit ftir Biochemie und Molekulare Biologic, Franklinstr. 29, D-1000 Berlin 10, Germany.
chains, a consequence of the amphidiploid nature of this species arising from interspecific hybridization from the diploid parents N. sylvestris and N. tomentosiformis [3]. Pinck et al. [4] have shown that the genome of N. sylvestris itself contains multiple small-subunit genes: their study of all transcripts argues for the possibility that only two families of genes are expressed in leaves of N. sylvestris. The amino acid sequence of the mature polypeptide is, however, not affected since the observed base differences in the coding region are silent. A Southern blot analysis of restriction fragments of all three Nicotiana species by Jamet et al. [5] has recently confirmed the existence of a multigene family as well as the progeny-progenitor relation as postulated by Gray et al. [3] already in 1974. Molecular biological studies of the rbcS gene revealed in recent years the existence of multigene families for this polypeptide present in many plant species. Thus, the rbcS gene is present in several copies in petunias, peas, wheat and soy beans with up to ten members for the latter [6--9]. The wheat genome contains more than ten rbcS gene copies [10] and duckweed (Lemna gibba) even thirteen [11]. The multigene families have been characterized ex-
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
clusively at the genomic or transcriptional level, translation products are in general less studied. Sheen and Bogorad [12] describe for maize three rbcS genes with two transcripts representing > 80% of the total rbcS-mRNA in green leaves. The authors investigated also the polypeptide pattern of the small subunit by isotope incorporation followed by two-dimensional g e l electrophoresis demonstrating the existence of two major distinct subunits with traces of 2 4 additional bands. These results are in good agreement with a recent proteinchemical sequence determination pointing to several amino acid heterogeneities which arise as the consequence of the expression of two (or more) genes [13]. Here we report for the small subunit from maize rubisco five isoforms which can be clearly distinguished by their isoelectric points; for the small subunit of spinach rubisco, for which hitherto no genomic analysis has been performed, two isoforms are observed. Materials and Methods
1,4-Dithioerythritol (DTE) was purchased from Biomol (Hamburg, Germany) and urea from Merck (Darmstadt, Germany), AcA44 ultrogel was obtained from LKB/Pharmacia (Freiburg, Germany).
centrifugation. Analysis of the precipitate showed that it mainly consisted of subunit isoform 2 (see Results). The soluble supernatant of the dialyzate containing the bulk small subunit was lyophilized and subjected to gel electrophoresis. Two-dimensional gel electrophoresis was essentially performed as described by O'Farrell [15]. Purified spinach rubisco was analyzed by twodimensional gel electrophoresis in toto omitting the isolation of the small subunit. The pH-gradient after one-dimensional isoelectric focusing was determined experimentally by slicing the gel rod into small segments and measuring their pH after equilibration in distilled water. The pl values of the isoforms were obtained by graphical regression analysis.
Automated solid phase sequencing The amino terminus of electrophoretical pure maize subunit isoform 2 was sequenced after on column coupling to aminopropyl glass viap-phenylenediisothiocyanate [16] by automated solid-phase Edman degradation with 4-N,N-dimethylaminoazobenzene-4 '-isothiocyanate phenylisothiocyanate as described earlier [17]. The first amino acid usually remains bound to the resin and is not identified. Results
Purification of rubisco Maize rubisco was purified according to Ren et al. [13]. Complete removal of phosphoenolpyruvate carboxylase was not attempted since this enzyme does not contain small subunits and its presence does not interfere with the preparation of rubisco small subunits. Spinach rubisco was prepared essentially as described by Vater and Salnikow [14]. Isolation and two-dimensional gel electrophoretical analysis of rubisco small subunits Purified maize rubisco was solubilized in 0.1 M Tris buffer (pH 8.5), containing 8 M urea and 100 mM DTE and after incubation for 30 min at room temperature applied to an AcA44 gel filtration column (2 x 100 cm) equilibrated and eluted with the same buffer containing, however, 4 M ui-ea and 50 mM DTE. Pooled fractions of rubisco small subunit were dialyzed against distilled water; routinely a small precipitate appeared which was removed by
Preparative &olation of the native small subunit of rub&co Citraconylation of total rubisco introducing cumulated negative charges on the protein surface leads to facile dissociation of the subunits [2]; this procedure is, however, not applicable when isoelectric points need to be determined. In order to obtain small subunits in their native state purified rubisco was dissociated in 8 M urea in the presence of DTE and subjected to gel filtration on AcA44 in the presence of 4 M urea yielding pure small subunit as judged by analytical SDS-gel electrophoresis. Two-dimensional analysis of native small subunits from rubisco The fractions containing the small subunit from maize rubisco were pooled and analyzed by twodimensional gel electrophoresis using the standard
method o f O'Farrell [15]. As seen in Fig. la the small subunit fraction o f maize rubisco resolves into five polypeptide bands which differ in their isoelectrical points; the graphical determination o f
these p/-values yields a range from p I = 7.4--5.2 for the five isoforms. Whereas isoforms 1 and 3 represent the major amount of the total subunit population, isoforms 2 and 4 are minor and isoform
Isoelectrofocusing-----
09
g..
O
§ t.._
09 09 09 09 "ID
K,
t) O >., O Cl_ I
t/3 C'3 t/3
Fig. 1. Two-dimensional gel electrophoresis of: (a) The small subunit mixture isolated from maize rubisco; the five isoforms are characterized by their p/-values. Left lane: molecular weight standards. (Marker proteins have been applied in the second dimension after isoelectrofocusing of the sample; the apparent multiplicity of marker proteins is due to accidental diffusion during application). (b) Purified spinach rubisco. SS, small subunit, LS, large subunit.
5 trace constituents. These results are in excellent agreement with those obtained by Sheen and Bogorad [12] by in vivo isotope labelling and explain the frequent occurrence of amino acid dimorphies observed during chemical sequencing of the maize small subunit. Basically, however, caution has to be applied in the interpretation of translation products excluding artifacts or post-translational modifications generating multiple polypeptide patterns. Thus, carbamylation of proteins by urea containing traces of cyanate can lead to multiple artifacts and is a well-known source of misinterpretation; dissociation of rubisco performed in 4 M guanidine chloride, however, yields the same isoelectric pattern (results not shown). Furthermore, it can be argued that the presence of Tris ions in concentrated urea solutions serves as an internal cyanate scavenger preventing unwanted carbamylation. In an attempt to characterize one of the multiple maize small subunits by amino terminal sequencing isoform 2 could be selectively recovered by its precipitation as a pure polypeptide due to its rather low solubility in water during dialysis of the total maize subunit pool. The following amino terminus was identified: 1X-Gln-Val-Trp-Pro-6Ala-Tyr-GlyAsn... The chemically determined sequence of the whole small subunit pool shows in pos. 6 of this amino terminus a dimorphy Ala/Ile [13], i.e. in this position both amino acids are observed. This means that in this publication sequencing has been performed on a mixture of two polypeptides possessing in this sequence position different amino acid residues. In isoform 2, however, in pos. 6 only Ala is found, Ile is clearly absent indicating that the separation of the small subunit isoforms as observed in two-dimensional gel electrophoresis is probably rather due to differences in amino acid sequence than posttranslational modifications which could also lead to pI differences and, consequently, to a complex isoelectrofocusing pattern. For comparison with maize rubisco the corresponding enzyme from spinach has been subjected to two-dimensional analysis. Here, again, the small subunit resolves into two isoelectrically different isoforms with pI values of 6.6 and 7.2, respectively. In Fig. lb in addition to the two major small subunit isoforms faint traces of two further polypeptide bands are visible. At present it is not clear, if
these traces are possibly artifacts or native trace constituents. Although the three-dimensional structure of spinach rubisco has been solved recently [18], to our knowledge this small subunit heterogeneity has never been described so far. Discussion
The gene expression of the small subunit multigene family of several plant rubisco's has been analyzed at the transcriptional level in detail. Thus, in peas transcriptional controls vary in a tissuespecific and light-regulated fashion [19,20] and transcript levels are regulated by phytochrom [21]. Of the eight small subunit genes present in the petunia multigene family one gene accounts for almost half of the total rbcS messenger RNA with distinctly lower levels for the residual genes and considerable variation for different plant organs [22]. A detailed analysis of the multigene family of the small subunit from tomato rubisco has recently been published by Sugita and Gruissem [23]; the authors characterized five genes which were all expressed in leaves with two specific transcripts representing about 60% of the total messenger RNA. Not only are certain genes of the gene family specifically transcribed, the whole transcription program appears to be organ-specific and is regulated by distinct phases of plant development. All these observations support strongly an organ-specific and light-induced expression for multigene families of the rubisco small subunit type at the transcriptional level [24]. In the present study the native small subunit of maize rubisco has been electrophoretically resolved into five isoforms of identical molecular weight possessing, however, different isoelectric points (pI = 7.4, 7.2, 6.5, 5.7 and 5.2). These results are in agreement with the gene expression studies of Sheen and Bogorad [12] who identify three rbeS genes with two major transcripts and observe two major polypeptides with traces of additional isoforms. Protein-chemical sequencing of the unresolved small subunit revealed several amino acid dimorphies along the polypeptide chain [13] pointing to the presence of a mixture of two (or more) slightly different isoforms. In an attempt to characterize one of the isoforms obtained in pure form by amino-
terminal sequencing a dimorphy found formerly in pos. 6 [13] had disappeared supporting the premise that the isoforms differ in their amino acid sequence. For definite proof, however, the isolation and characterization of all isoforms by comparative techniques like peptide mapping will be required. In addition to maize, also for spinach two major isoforms with p l = 6.6 and 7.2 were identified. Thus, the small subunit multiplicity observed in this study mirrors closely the nmltigene expression at the translational level and matches the framework obtained by analysis of gene transcripts. Since the isoforms were prepared from whole plant material with undefined developmental characteristics, the distribution pattern obtained represents a rather tinfocussed snapshot ofmultigene expression. The twodimensional technique as described in this study, however, should permit a detailed quantitative study of multigene expression at the translational level as function-of organ specificity, light regulation and developmental stages. Acknowledgements The authors thank G. Haeselbarth for expert help in automated solid phase sequencing.
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References 1
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S.D. Kung, K. Sakano and S.G. Wildman, Multiple peptide composition of the large and small subunits of Nicotiana tabacum fraction I protein ascertained by fingerprinting and electrofocusing. Biochim. Biophys. Acta, 365 (1974) 138--147. K.-D. M/iller, J. Salnikow and J. Vater, Amino acid sequence of the small subunit of D-ribulosebisphosphate carboxylase/oxygenase from Nieotiana tabaeum. Biochim. Biophys. Acta, 742 (1983) 78--83. J.C. Gray, S.D. Kung and S.-G. Wildman, Origin of Nicotiana tabaeum L. detected by polypeptide composition of fraction I protein. Nature, 252 (1974) 226~22. M. Pinck, J.-M. Dore, E. Guilley, A. Durr, L. Pinck, L. Hirth and J. Fleck, A simple gene expression system for the small subunit of ribulose bisphosphate carboxylase in leaves of Nieotiana sylvestris. Plant Mol. Biol., 7 (1986) 301--309. E. Jamet, A. Durr and J. Fleck, Absence of some truncated genes in the amphidiploid Nicotiana tabaeum. Gene, 59 (1987) 213--221. P. Duusmuir, S. Smith and J. Bedbrook, A number of different nuclear genes for the small subunit of RuBPCase are transcribed in petunia. Nucl. Acids Res., 11 (1983) 4177~183.
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A.R. Cashmore, Nuclear genes encoding the small subunit of ribulose-l,5-bisphosphate carboxylase, in: T. Kosuge, C.P. Meredith and A. Hollaender (Eds.), Genetic Engineering of Plants, Plenum Press, New York, 1983, pp. 29--38. G. Coruzzi, R. Broglie, A. Cashmore and N.H. Chua, Nucleotide sequence of two pea cDNA clones encoding the small subunit of ribulose-l,5-bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptide. J. Biol. Chem., 258 (1983) 1399--1402. S.L. Berry-Lowe, T.D. McKnight, D.M. Shaw and R.B. Meagher, The nucleotide sequence, expression and evolution of one member of a multigene family encoding the small subunit of ribulose-l,5-bisphosphate carboxylase in soybean. J. Mol. Appl. Genet., 2 (1982) 483~498. S.M. Smith, J. Bedbrook and J. Speirs, Characterisation of three cDNA clones encoding different mRNAs for the precursor to the small subunit of wheat ribulosebisphosphate carboxylase. Nucl. Acids Res., 11 (1983) 8719--8734. C.F. Wimpee, Organization and expression of lightregulated genes in Lemma gibba L. G-3, Ph.D. thesis, University of California, Los Angeles; 1984. J.-Y. Sheen and L. Bogorad, Expression of the ribulose-l,5-bisphosphate carboxylase large subunit gene and three small subunit genes in two cell types of maize leaves. EMBO J., 5 (1986) 3417--3422. L. Ren, J. Salnikow and J. Vater, Ribulose-l,5-bisphosphate carboxylase/oxygenase from Zea mays: amino-acid sequence of the small subunit. Biol. Chem. Hoppe-Seyler, 369 (1988) 609--615. J. Vater and J. Salnikow, Identification of two binding sites of the D-ribulose-l,5-bisphosphate carboxylase/oxygenase from spinach for D-ribulose 1,5-bisphosphate and effectors of the carboxylation reaction. Arch. Biochem. Biophys., 194 (1979) 190--197. P.H. O'Farrell, High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem., 250 (1975) 4007~4021. J. Salnikow, Automated solid-phase microsequencing using DAB1TC, on-column immobilization of proteins, in: B. Wittmann-Liebold et al. (Eds.), Advanced Methods in Protein Microsequence Analysis, Springer-Verlag, Berlin, Heidelberg, 1986, pp. 108--116. J. Salnikow, A. Lehmann and B. Wittmann-Liebold, Improved automated solid-phase microsequencing of peptides using DABITC. Anal. Biochem., 117 (1981) 433--442. I. Andersson, S. Knight, G. Schneider, Y. Lindqvist, T. Lundqvist, C.-1. Br~inden and G.H. Lorimer, Crystal structure of the active site of ribulose-bisphosphate carboxylase. Nature, 337 (1989) 229--234. G. Coruzzi, R. Broglie, C. Edwards and N.H. Chua, Tissuespecific and light-related expression of a pea nuclear gene encoding the small subunit of ribulose-l,5-bisphosphate carboxyase. EMBO J., 3 (1984) 1671--1679. R. Broglie, G. Coruzzi, R.T. Fraley, S.G. Rogers, R.B. Horsch, J.G. Niedermeyer, C.L. Fink, J.S. Flick and N.-H. Chua, Light-regulated expression of a pea
ribulose-l,5-bisphosphate carboxylase small subunit gene in transformed plant-cells. Science, 224 (1984) 838--843. 21 R. Fluhr, and N.-H. Chua, Developmental regulation of two genes encoding ribulose-bisphosphate earboxylase small subunit in pea and transgenic petunia plant: phytochrome response and blue-light induction. Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 2358--2362. 22 C. Dean, P. Van den Elzen, S. Tamaki, P. Dunsmuir and J. Bedbrook, Differential expression of the eight genes of
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the petunia ribulose bisphosphate carboxylase small subunit multi-gene family. EMBO J., 4 (1985) 3055--3061. M. Sugita and W. Gruissem, Developmental, organ-specific and light-dependent expression of the tomato ribulose-l,5-bisphosphate carboxylase small subunit gene family. Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7104--7108. C. Dean, E. Pichersky and P. Dunsmuir, Structure, evolution and regulation of RbcS genes in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol., 40 (1989) 415---439.
1
Multiple forms of the small subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase in maize and spinach Lifen Ren, Johann Salnikow and Joachim Vater Technische Universitdt Berlin, Institiit fiir Biochemie und Molekulare Biologic, Franklinstr. 29, D-IO00 Berlin 10 (Germany) (Received June 1st, 1990; revision received October 22nd, 1990; accepted October 22nd, 1990)
Native small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) from maize has been electrophoretically resolved into five isoforms of identical molecular weight possessing, however, different isoelectric points (pl = 7.4, 7.2, 6.5, 5.7 and 5.2). Gene expression studies (Sheen and Bogorad, EMBO J., 5 (1986) 3417) and proteinchemical sequencing (Ren et al., Biol. Chem. HoppeSeyler, 369 (1988) 609) suggest that the observed isoforms have slightly different amino acid sequences as a result of a multigene expression. For spinach rubisco the native small subunit resolves into two isoforms with p l = 6.6 and 7.2.
Key words: ribulose-l,5-bisphosphate; carboxylase/oxygenase; isoforms
Introduction
Ribulose-l,5-bisphosphate carboxylase/oxygenase ('rubisco') as the central enzyme of photosynthetic carbon dioxide fixation is present in all plants and photosynthetic microorganisms. Its architecture in plants is formed by the assembly of eight large subunits carrying the catalytic active residues and eight small subunits. Whereas the large subunits are plastome coded, the genes for the small subunits (rbcS) are located in the nucleus and translated into precursor polypeptides carrying an amino-terminal extension which serves as the recognition signal for the chloroplast organelle. One of the earliest examples for small subunit multiplicity concerns the tomato plant; Kung et al. [1] demonstrated in 1974 by isoelectric focusing and fingerprinting two discernible polypeptide chains for the small subunit from Nicotiana tabacum, M/iller et al. [2] confirmed by protein chemical sequencing the existence of two slightly different polypeptide Correspondence to: Johann Salnikow, Technische Universit/it Berlin, Instittit ftir Biochemie und Molekulare Biologic, Franklinstr. 29, D-1000 Berlin 10, Germany.
chains, a consequence of the amphidiploid nature of this species arising from interspecific hybridization from the diploid parents N. sylvestris and N. tomentosiformis [3]. Pinck et al. [4] have shown that the genome of N. sylvestris itself contains multiple small-subunit genes: their study of all transcripts argues for the possibility that only two families of genes are expressed in leaves of N. sylvestris. The amino acid sequence of the mature polypeptide is, however, not affected since the observed base differences in the coding region are silent. A Southern blot analysis of restriction fragments of all three Nicotiana species by Jamet et al. [5] has recently confirmed the existence of a multigene family as well as the progeny-progenitor relation as postulated by Gray et al. [3] already in 1974. Molecular biological studies of the rbcS gene revealed in recent years the existence of multigene families for this polypeptide present in many plant species. Thus, the rbcS gene is present in several copies in petunias, peas, wheat and soy beans with up to ten members for the latter [6--9]. The wheat genome contains more than ten rbcS gene copies [10] and duckweed (Lemna gibba) even thirteen [11]. The multigene families have been characterized ex-
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
clusively at the genomic or transcriptional level, translation products are in general less studied. Sheen and Bogorad [12] describe for maize three rbcS genes with two transcripts representing > 80% of the total rbcS-mRNA in green leaves. The authors investigated also the polypeptide pattern of the small subunit by isotope incorporation followed by two-dimensional g e l electrophoresis demonstrating the existence of two major distinct subunits with traces of 2 4 additional bands. These results are in good agreement with a recent proteinchemical sequence determination pointing to several amino acid heterogeneities which arise as the consequence of the expression of two (or more) genes [13]. Here we report for the small subunit from maize rubisco five isoforms which can be clearly distinguished by their isoelectric points; for the small subunit of spinach rubisco, for which hitherto no genomic analysis has been performed, two isoforms are observed. Materials and Methods
1,4-Dithioerythritol (DTE) was purchased from Biomol (Hamburg, Germany) and urea from Merck (Darmstadt, Germany), AcA44 ultrogel was obtained from LKB/Pharmacia (Freiburg, Germany).
centrifugation. Analysis of the precipitate showed that it mainly consisted of subunit isoform 2 (see Results). The soluble supernatant of the dialyzate containing the bulk small subunit was lyophilized and subjected to gel electrophoresis. Two-dimensional gel electrophoresis was essentially performed as described by O'Farrell [15]. Purified spinach rubisco was analyzed by twodimensional gel electrophoresis in toto omitting the isolation of the small subunit. The pH-gradient after one-dimensional isoelectric focusing was determined experimentally by slicing the gel rod into small segments and measuring their pH after equilibration in distilled water. The pl values of the isoforms were obtained by graphical regression analysis.
Automated solid phase sequencing The amino terminus of electrophoretical pure maize subunit isoform 2 was sequenced after on column coupling to aminopropyl glass viap-phenylenediisothiocyanate [16] by automated solid-phase Edman degradation with 4-N,N-dimethylaminoazobenzene-4 '-isothiocyanate phenylisothiocyanate as described earlier [17]. The first amino acid usually remains bound to the resin and is not identified. Results
Purification of rubisco Maize rubisco was purified according to Ren et al. [13]. Complete removal of phosphoenolpyruvate carboxylase was not attempted since this enzyme does not contain small subunits and its presence does not interfere with the preparation of rubisco small subunits. Spinach rubisco was prepared essentially as described by Vater and Salnikow [14]. Isolation and two-dimensional gel electrophoretical analysis of rubisco small subunits Purified maize rubisco was solubilized in 0.1 M Tris buffer (pH 8.5), containing 8 M urea and 100 mM DTE and after incubation for 30 min at room temperature applied to an AcA44 gel filtration column (2 x 100 cm) equilibrated and eluted with the same buffer containing, however, 4 M ui-ea and 50 mM DTE. Pooled fractions of rubisco small subunit were dialyzed against distilled water; routinely a small precipitate appeared which was removed by
Preparative &olation of the native small subunit of rub&co Citraconylation of total rubisco introducing cumulated negative charges on the protein surface leads to facile dissociation of the subunits [2]; this procedure is, however, not applicable when isoelectric points need to be determined. In order to obtain small subunits in their native state purified rubisco was dissociated in 8 M urea in the presence of DTE and subjected to gel filtration on AcA44 in the presence of 4 M urea yielding pure small subunit as judged by analytical SDS-gel electrophoresis. Two-dimensional analysis of native small subunits from rubisco The fractions containing the small subunit from maize rubisco were pooled and analyzed by twodimensional gel electrophoresis using the standard
method o f O'Farrell [15]. As seen in Fig. la the small subunit fraction o f maize rubisco resolves into five polypeptide bands which differ in their isoelectrical points; the graphical determination o f
these p/-values yields a range from p I = 7.4--5.2 for the five isoforms. Whereas isoforms 1 and 3 represent the major amount of the total subunit population, isoforms 2 and 4 are minor and isoform
Isoelectrofocusing-----
09
g..
O
§ t.._
09 09 09 09 "ID
K,
t) O >., O Cl_ I
t/3 C'3 t/3
Fig. 1. Two-dimensional gel electrophoresis of: (a) The small subunit mixture isolated from maize rubisco; the five isoforms are characterized by their p/-values. Left lane: molecular weight standards. (Marker proteins have been applied in the second dimension after isoelectrofocusing of the sample; the apparent multiplicity of marker proteins is due to accidental diffusion during application). (b) Purified spinach rubisco. SS, small subunit, LS, large subunit.
5 trace constituents. These results are in excellent agreement with those obtained by Sheen and Bogorad [12] by in vivo isotope labelling and explain the frequent occurrence of amino acid dimorphies observed during chemical sequencing of the maize small subunit. Basically, however, caution has to be applied in the interpretation of translation products excluding artifacts or post-translational modifications generating multiple polypeptide patterns. Thus, carbamylation of proteins by urea containing traces of cyanate can lead to multiple artifacts and is a well-known source of misinterpretation; dissociation of rubisco performed in 4 M guanidine chloride, however, yields the same isoelectric pattern (results not shown). Furthermore, it can be argued that the presence of Tris ions in concentrated urea solutions serves as an internal cyanate scavenger preventing unwanted carbamylation. In an attempt to characterize one of the multiple maize small subunits by amino terminal sequencing isoform 2 could be selectively recovered by its precipitation as a pure polypeptide due to its rather low solubility in water during dialysis of the total maize subunit pool. The following amino terminus was identified: 1X-Gln-Val-Trp-Pro-6Ala-Tyr-GlyAsn... The chemically determined sequence of the whole small subunit pool shows in pos. 6 of this amino terminus a dimorphy Ala/Ile [13], i.e. in this position both amino acids are observed. This means that in this publication sequencing has been performed on a mixture of two polypeptides possessing in this sequence position different amino acid residues. In isoform 2, however, in pos. 6 only Ala is found, Ile is clearly absent indicating that the separation of the small subunit isoforms as observed in two-dimensional gel electrophoresis is probably rather due to differences in amino acid sequence than posttranslational modifications which could also lead to pI differences and, consequently, to a complex isoelectrofocusing pattern. For comparison with maize rubisco the corresponding enzyme from spinach has been subjected to two-dimensional analysis. Here, again, the small subunit resolves into two isoelectrically different isoforms with pI values of 6.6 and 7.2, respectively. In Fig. lb in addition to the two major small subunit isoforms faint traces of two further polypeptide bands are visible. At present it is not clear, if
these traces are possibly artifacts or native trace constituents. Although the three-dimensional structure of spinach rubisco has been solved recently [18], to our knowledge this small subunit heterogeneity has never been described so far. Discussion
The gene expression of the small subunit multigene family of several plant rubisco's has been analyzed at the transcriptional level in detail. Thus, in peas transcriptional controls vary in a tissuespecific and light-regulated fashion [19,20] and transcript levels are regulated by phytochrom [21]. Of the eight small subunit genes present in the petunia multigene family one gene accounts for almost half of the total rbcS messenger RNA with distinctly lower levels for the residual genes and considerable variation for different plant organs [22]. A detailed analysis of the multigene family of the small subunit from tomato rubisco has recently been published by Sugita and Gruissem [23]; the authors characterized five genes which were all expressed in leaves with two specific transcripts representing about 60% of the total messenger RNA. Not only are certain genes of the gene family specifically transcribed, the whole transcription program appears to be organ-specific and is regulated by distinct phases of plant development. All these observations support strongly an organ-specific and light-induced expression for multigene families of the rubisco small subunit type at the transcriptional level [24]. In the present study the native small subunit of maize rubisco has been electrophoretically resolved into five isoforms of identical molecular weight possessing, however, different isoelectric points (pI = 7.4, 7.2, 6.5, 5.7 and 5.2). These results are in agreement with the gene expression studies of Sheen and Bogorad [12] who identify three rbeS genes with two major transcripts and observe two major polypeptides with traces of additional isoforms. Protein-chemical sequencing of the unresolved small subunit revealed several amino acid dimorphies along the polypeptide chain [13] pointing to the presence of a mixture of two (or more) slightly different isoforms. In an attempt to characterize one of the isoforms obtained in pure form by amino-
terminal sequencing a dimorphy found formerly in pos. 6 [13] had disappeared supporting the premise that the isoforms differ in their amino acid sequence. For definite proof, however, the isolation and characterization of all isoforms by comparative techniques like peptide mapping will be required. In addition to maize, also for spinach two major isoforms with p l = 6.6 and 7.2 were identified. Thus, the small subunit multiplicity observed in this study mirrors closely the nmltigene expression at the translational level and matches the framework obtained by analysis of gene transcripts. Since the isoforms were prepared from whole plant material with undefined developmental characteristics, the distribution pattern obtained represents a rather tinfocussed snapshot ofmultigene expression. The twodimensional technique as described in this study, however, should permit a detailed quantitative study of multigene expression at the translational level as function-of organ specificity, light regulation and developmental stages. Acknowledgements The authors thank G. Haeselbarth for expert help in automated solid phase sequencing.
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References 1
2
3
4
5
6
S.D. Kung, K. Sakano and S.G. Wildman, Multiple peptide composition of the large and small subunits of Nicotiana tabacum fraction I protein ascertained by fingerprinting and electrofocusing. Biochim. Biophys. Acta, 365 (1974) 138--147. K.-D. M/iller, J. Salnikow and J. Vater, Amino acid sequence of the small subunit of D-ribulosebisphosphate carboxylase/oxygenase from Nieotiana tabaeum. Biochim. Biophys. Acta, 742 (1983) 78--83. J.C. Gray, S.D. Kung and S.-G. Wildman, Origin of Nicotiana tabaeum L. detected by polypeptide composition of fraction I protein. Nature, 252 (1974) 226~22. M. Pinck, J.-M. Dore, E. Guilley, A. Durr, L. Pinck, L. Hirth and J. Fleck, A simple gene expression system for the small subunit of ribulose bisphosphate carboxylase in leaves of Nieotiana sylvestris. Plant Mol. Biol., 7 (1986) 301--309. E. Jamet, A. Durr and J. Fleck, Absence of some truncated genes in the amphidiploid Nicotiana tabaeum. Gene, 59 (1987) 213--221. P. Duusmuir, S. Smith and J. Bedbrook, A number of different nuclear genes for the small subunit of RuBPCase are transcribed in petunia. Nucl. Acids Res., 11 (1983) 4177~183.
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