Photosynthesis Research 16:117-139 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands
Minireview
Organization and expression of the genes encoding ribulose-l,5-bisphosphate carboxylase in higher plants T H I A N D A M A N Z A R A & WILHELM GRUISSEM Department of Botany, University of California, Berkeley, CA 94720, USA Received 13 October 1987; accepted 3 December 1987)
Key words: higher plants, rbcL, rbcS, ribulose-l,5-bisphosphate carboxylase, Rubisco, SSU
Introduction
The enzyme ribulose-l,5-bisphosphate carboxylase (Rubisco) catalyzes the first step in photosynthetic CO2 fixation in higher plants. Rubisco is a chloroplast enzyme consisting of 16 subunits (Jensen and Bahr 1977), eight large subunits of molecular weight approximately 55,000, and eight small subunits of molecular weight approximately 14,000 (Blair and Ellis 1973). The large subunit of the enzyme contains the catalytic site (Lorimer and Miziorko 1980, Lorimer 1981), is encoded by the chloroplast genome (Chan and Wildman 1972, Coen et al. 1977), and is synthesized on chloroplast ribosomes (Blair and Ellis 1973). The small subunit is encoded by the nuclear genome as a small multigene family of 5 to 15 members in most higher plants (Berry-Lowe et al. 1982, Broglie et al. 1983, Coruzzi et al. 1983, Dunsmuir et al. 1983, Wimpee et al. 1983). The small subunit protein is synthesized on cytoplasmic ribosomes as a larger precursor polypeptide which is cleaved to the mature form during import into the chloroplasts, where the holoenzyme is then assembled (Kawashimia and Wildman 1972, Highfield and Ellis 1978, reviewed by Ellis 1981). In the past several years workers in a number of laboratories have been engaged in studying the molecular basis of Rubisco gene expression. Because Rubisco is a highly abundant enzyme in plants, its biochemical properties were characterized early on, and the protein was purified and partially sequenced. This facilitated the identification of the genes for the large subunit (rbcL) and the small subunit (rbcS), which were among the first chloroplast and nuclear genes, respectively, to be cloned from higher plants. Since then, rbcL and rbcS have been sequenced, and their expression has
118 been extensively characterized in several higher plants. The expression of rbcS and rbcL provides an important model system to study the developmental and light regulation of genes for photosynthetic proteins. For example, our understanding of rbcL expression will be applicable to the broader question of how chloroplast gene expression is regulated as a whole. Likewise, understanding the regulation of expression of the rbcS gene family will provide important information for the more general question of how nuclear gene expression is regulated, and in particular, how expression of nearly identical members of a multigene family can be regulated differentially. Finally, learning how approximately equimolar amounts of small and large subunit polypeptides are synthesized, given the fact that the gene dosage of rbcS and rbcL in a plant cell are vastly different, will help us understand how gene expression is coordinated between the nuclear and chloroplast compartments. In this review we will compare the structure and organization of the rbcL and rbcS genes from a number of higher plants. In addition, we will review what is known about the in vivo expression of rbcS and rbcL at the mRNA level, and will examine how expression of these genes is regulated. We will emphasize the work on tomato that has recently been completed in our laboratory, especially with regard to the rbcS gene family. We will compare and contrast the in vivo expression data for the rbcS genes of tomato, petunia, and pea, which represent the higher plants for which detailed information is available on the expression of the individual rbcS genes. In an effort to identify upstream sequences which may influence rbcS gene expression, we will compare the 5' flanking regions of all rbcS genes which have been sequenced from dicotyledonous plants, and will discuss experiments which have addressed the question of the functional significance of rbcS conserved upstream sequences.
Structure and organization of the rbcL and rbcS genes
Evidence that the rbcL gene was chloroplast encoded and that rbcS was nuclear encoded initially came from the results of genetic crosses (Chan and Wildman 1972, Kawashima and Wildman 1972), and from studies in which the large subunit polypeptide was synthesized in vitro using isolated chloroplasts (Blair and Ellis 1973) or purified chloroplast RNA (Hartley et al. 1975, Sagher et al. 1976). The first rbcL gene isolated from a higher plant was cloned from maize chloroplast DNA by Coen et al. (1977), who used a rabbit-reticulocyte in vitro transcription-translation system to screen cloned maize chloroplast DNA fragments for production of a large subunit-sized
119 polypeptide. The identity of the polypeptide product produced by the cloned DNA was then verified serologically and by proteolytic cleavage. Since that time, rbcL has been mapped on the chloroplast genome in a number of higher plants (compiled by Crouse et al. 1985). In chloroplast genomes which have the typical inverted repeat structure, the rbcL gene is located in the large single copy region next to, and of opposite polarity from the polycistronic genes encoding the beta- and epsilon-subunits of the chloroplast ATPase (atpB/E). The rbcL gene has been sequenced in spinach (Zurawski et al. 1981), petunia (Aldrich et al. 1986a), alfalfa (Aldrich et al. 1986b), maize (McIntosh et al. 1980), pea (Zurawski et al. 1986), tobacco (Shinozaki and Sugiura 1982), barley (Zurawski et al. 1984), Nicotiana otophora and N. acuminata (Lin et al. 1986), and has been partially sequenced in tomato (Manzara, Chonoles-Imlay and Gruissem, unpublished). In all higher plants studied to date, the rbcL coding sequence is continuous, approximately 1.4 kb in length, and encodes a polypeptide of approximately 475 amino acids. The coding sequences are highly conserved between rbcL genes of different species, with the majority of nucleotide changes occurring at the 3' ends of the genes. In addition, many of the base changes are silent, resulting in even higher homology between the amino acid sequences. For example, the homology between the rbcL nucleotide sequences of maize versus spinach is 84%, while the homology between the corresponding amino acid sequences is 90% (Zurawski et al. 1981). In closely related species, such as petunia and tobacco, the amino acid sequences are 97% conserved, and the nucleotide sequences of the 5' and 3' flanking regions are also highly homologous (Aldrich et al. 1986a). In less related species, the nucleotide sequence conservation between the 5' and 3' flanking regions is considerably lower, although functional domains are conserved (Zurawski and Clegg 1987). Evolutionary relationships based on rbcL sequence data from the 5' flanking and coding regions of a number of higher plants have been determined by Ritland and Clegg (1987). In all higher plants studied to date, the small subunit is encoded as a multigene family in the nuclear genome. For example, the rbcS multigene families contain at least ten members in soybean (Berry-Lowe et al. 1982), five members in pea (Coruzzi et al. 1983), eight members in petunia (Dunsmuir et al. 1983), thirteen members in duckweed (Wimpee et al. 1983), and five members in tomato (Sugita et al. 1987). The first rbcS gene was identified from a pea cDNA library by Bedbrook et al. (1980), who selected clones containing rbcS by hybridization to polyA mRNA fractions differentially enriched for rbcS mRNA, either by size fractionation or by preparation of mRNA from light versus dark grown pea leaves. The identity of the putative rbcS cDNA clones was then verified by DNA sequence analysis which
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Minireview
Organization and expression of the genes encoding ribulose-l,5-bisphosphate carboxylase in higher plants T H I A N D A M A N Z A R A & WILHELM GRUISSEM Department of Botany, University of California, Berkeley, CA 94720, USA Received 13 October 1987; accepted 3 December 1987)
Key words: higher plants, rbcL, rbcS, ribulose-l,5-bisphosphate carboxylase, Rubisco, SSU
Introduction
The enzyme ribulose-l,5-bisphosphate carboxylase (Rubisco) catalyzes the first step in photosynthetic CO2 fixation in higher plants. Rubisco is a chloroplast enzyme consisting of 16 subunits (Jensen and Bahr 1977), eight large subunits of molecular weight approximately 55,000, and eight small subunits of molecular weight approximately 14,000 (Blair and Ellis 1973). The large subunit of the enzyme contains the catalytic site (Lorimer and Miziorko 1980, Lorimer 1981), is encoded by the chloroplast genome (Chan and Wildman 1972, Coen et al. 1977), and is synthesized on chloroplast ribosomes (Blair and Ellis 1973). The small subunit is encoded by the nuclear genome as a small multigene family of 5 to 15 members in most higher plants (Berry-Lowe et al. 1982, Broglie et al. 1983, Coruzzi et al. 1983, Dunsmuir et al. 1983, Wimpee et al. 1983). The small subunit protein is synthesized on cytoplasmic ribosomes as a larger precursor polypeptide which is cleaved to the mature form during import into the chloroplasts, where the holoenzyme is then assembled (Kawashimia and Wildman 1972, Highfield and Ellis 1978, reviewed by Ellis 1981). In the past several years workers in a number of laboratories have been engaged in studying the molecular basis of Rubisco gene expression. Because Rubisco is a highly abundant enzyme in plants, its biochemical properties were characterized early on, and the protein was purified and partially sequenced. This facilitated the identification of the genes for the large subunit (rbcL) and the small subunit (rbcS), which were among the first chloroplast and nuclear genes, respectively, to be cloned from higher plants. Since then, rbcL and rbcS have been sequenced, and their expression has
118 been extensively characterized in several higher plants. The expression of rbcS and rbcL provides an important model system to study the developmental and light regulation of genes for photosynthetic proteins. For example, our understanding of rbcL expression will be applicable to the broader question of how chloroplast gene expression is regulated as a whole. Likewise, understanding the regulation of expression of the rbcS gene family will provide important information for the more general question of how nuclear gene expression is regulated, and in particular, how expression of nearly identical members of a multigene family can be regulated differentially. Finally, learning how approximately equimolar amounts of small and large subunit polypeptides are synthesized, given the fact that the gene dosage of rbcS and rbcL in a plant cell are vastly different, will help us understand how gene expression is coordinated between the nuclear and chloroplast compartments. In this review we will compare the structure and organization of the rbcL and rbcS genes from a number of higher plants. In addition, we will review what is known about the in vivo expression of rbcS and rbcL at the mRNA level, and will examine how expression of these genes is regulated. We will emphasize the work on tomato that has recently been completed in our laboratory, especially with regard to the rbcS gene family. We will compare and contrast the in vivo expression data for the rbcS genes of tomato, petunia, and pea, which represent the higher plants for which detailed information is available on the expression of the individual rbcS genes. In an effort to identify upstream sequences which may influence rbcS gene expression, we will compare the 5' flanking regions of all rbcS genes which have been sequenced from dicotyledonous plants, and will discuss experiments which have addressed the question of the functional significance of rbcS conserved upstream sequences.
Structure and organization of the rbcL and rbcS genes
Evidence that the rbcL gene was chloroplast encoded and that rbcS was nuclear encoded initially came from the results of genetic crosses (Chan and Wildman 1972, Kawashima and Wildman 1972), and from studies in which the large subunit polypeptide was synthesized in vitro using isolated chloroplasts (Blair and Ellis 1973) or purified chloroplast RNA (Hartley et al. 1975, Sagher et al. 1976). The first rbcL gene isolated from a higher plant was cloned from maize chloroplast DNA by Coen et al. (1977), who used a rabbit-reticulocyte in vitro transcription-translation system to screen cloned maize chloroplast DNA fragments for production of a large subunit-sized
119 polypeptide. The identity of the polypeptide product produced by the cloned DNA was then verified serologically and by proteolytic cleavage. Since that time, rbcL has been mapped on the chloroplast genome in a number of higher plants (compiled by Crouse et al. 1985). In chloroplast genomes which have the typical inverted repeat structure, the rbcL gene is located in the large single copy region next to, and of opposite polarity from the polycistronic genes encoding the beta- and epsilon-subunits of the chloroplast ATPase (atpB/E). The rbcL gene has been sequenced in spinach (Zurawski et al. 1981), petunia (Aldrich et al. 1986a), alfalfa (Aldrich et al. 1986b), maize (McIntosh et al. 1980), pea (Zurawski et al. 1986), tobacco (Shinozaki and Sugiura 1982), barley (Zurawski et al. 1984), Nicotiana otophora and N. acuminata (Lin et al. 1986), and has been partially sequenced in tomato (Manzara, Chonoles-Imlay and Gruissem, unpublished). In all higher plants studied to date, the rbcL coding sequence is continuous, approximately 1.4 kb in length, and encodes a polypeptide of approximately 475 amino acids. The coding sequences are highly conserved between rbcL genes of different species, with the majority of nucleotide changes occurring at the 3' ends of the genes. In addition, many of the base changes are silent, resulting in even higher homology between the amino acid sequences. For example, the homology between the rbcL nucleotide sequences of maize versus spinach is 84%, while the homology between the corresponding amino acid sequences is 90% (Zurawski et al. 1981). In closely related species, such as petunia and tobacco, the amino acid sequences are 97% conserved, and the nucleotide sequences of the 5' and 3' flanking regions are also highly homologous (Aldrich et al. 1986a). In less related species, the nucleotide sequence conservation between the 5' and 3' flanking regions is considerably lower, although functional domains are conserved (Zurawski and Clegg 1987). Evolutionary relationships based on rbcL sequence data from the 5' flanking and coding regions of a number of higher plants have been determined by Ritland and Clegg (1987). In all higher plants studied to date, the small subunit is encoded as a multigene family in the nuclear genome. For example, the rbcS multigene families contain at least ten members in soybean (Berry-Lowe et al. 1982), five members in pea (Coruzzi et al. 1983), eight members in petunia (Dunsmuir et al. 1983), thirteen members in duckweed (Wimpee et al. 1983), and five members in tomato (Sugita et al. 1987). The first rbcS gene was identified from a pea cDNA library by Bedbrook et al. (1980), who selected clones containing rbcS by hybridization to polyA mRNA fractions differentially enriched for rbcS mRNA, either by size fractionation or by preparation of mRNA from light versus dark grown pea leaves. The identity of the putative rbcS cDNA clones was then verified by DNA sequence analysis which
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