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Molecular cloning and expression of the large subunit of ADP-glucose pyrophosphorylase from barley (Hordeum vulgare) leaves
Gene 189 (1997) 79–82
Molecular cloning and expression of the large subunit of ADP-glucose pyrophosphorylase from barley (Hordeum vulgare) leaves Klaus Eimert a,1, Cheng Luo a, Annabelle De´jardin a, Per Villand a,b, Tine Thorbjørnsen b,2, Leszek A. Kleczkowski a,* a Department of Plant Physiology, Umea˚ University, 901-87 Umea˚, Sweden b Plant Molecular Biology Laboratory, Agricultural University of Norway, 1432 Aas, Norway Received 30 August 1996; revised 28 October 1996; accepted 29 October 1996; Received by J. Wild
Abstract A cDNA clone, blpl14, corresponding to the large subunit of ADP-glucose pyrophosphorylase (AGPase), has been isolated from a cDNA library prepared from leaves of barley (Hordeum vulgare L.). An open reading frame encodes a protein of 503 aa, with a calculated molecular weight of 54 815. The derived aa sequence contains a putative transit peptide sequence, required for targeting to plastids, and has a highly conserved positioning of critical Lys residues that are believed to be involved in effector binding. The derived aa sequence shows 97% identity with the corresponding protein from wheat, but only 36% identity with AGPase from E. coli. The blpl14 gene is expressed predominantly in leaves and to a lesser degree in seed endosperm, but not roots, of barley. Keywords: Chloroplast; Endosperm; Starch; Sucrose; Transit peptide
1. Introduction ADP-glucose pyrophosphorylase (AGPase) is the key enzyme of starch biosynthesis in all plants and bacteria. In plants, the enzyme is composed of two subunit types, encoded by different genes (Morell et al., 1987; Kleczkowski et al., 1991; Nelson and Pan, 1995). In barley, at least two isozymes of AGPase have been described, one in seed endosperm and the other – in leaves ( Kleczkowski et al., 1993). The isozymes differ in their kinetic properties and regulation ( Kleczkowski et al., 1993), and are localized in different cell compartments ( Villand and Kleczkowski, 1994; Thorbjørnsen et al., 1996a,b). Although several cDNA sequences for plant AGPase * Corresponding author. Tel. +46 90 167781; Fax +46 90 166676; e-mail: [email protected] 1 Current address: Department of Botany, Geisenheim Research Centre, 65366 Geisenheim, Germany. 2 Current address: Plant Biochemistry Laboratory, Royal Veterinary and Agricultural University, Thorvaldsens vej 40, Copenhagen, Denmark. Abbreviations: aa, amino acid(s); AGPase, ADP-glucose pyrophosphorylase; nt, nucleotide(s); ORF, open reading frame; UTR, untranslated region(s). 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 8 37 - 2
have already been published (summarized in La Cognata et al., 1995), there are only few reports describing isolation of cDNA(s) for the large subunit of the leaf form of the enzyme (Olive et al., 1989; Villand et al., 1992a, 1993; La Cognata et al., 1995). Previously, we have used a PCR technique to amplify several partial (approx. 550 nt) cDNAs corresponding to small and large subunits of AGPase from various plant tissues ( Villand et al., 1992a, 1993), including leaves. These PCR products have proven to be very useful as genespecific probes to isolate full length cDNAs, genomic clones and for expression studies in barley ( Villand et al., 1992b; Thorbjørnsen et al., 1996b). In the present study, isolation of a cDNA encoding ORF of the leaf form of the large subunit of barley AGPase is reported, along with characterization of its expression in barley tissues.
2. Experimental and discussion 2.1. Cloning of blpl14 The isolation of cDNA encoding AGPase was carried out by screening of a cDNA library prepared from
80
K. Eimert et al. / Gene 189 (1997) 79–82
mRNA isolated from leaves (3 weeks old ) of barley (Hordeum vulgare L. cv. Bomi). The library was custommade by Clontech (Palo Alto, CA, USA) using the l ZAPII system. A 550-nt blpl cDNA clone, previously isolated by PCR amplification on total barley leaf mRNA ( Villand et al., 1992a), was used as the hybridization probe. A putative full-length clone, blpl14, was sequenced on both strands using 32P-end-labelled custom-made primers (fmol DNA Sequencing, Promega, Madison, WI, USA).
2.2. Nucleotide and deduced amino-acid sequences A cDNA clone, blpl14 (GenBank accession No. U66876), encoding the large subunit of AGPase has been isolated from a barley leaf cDNA library. The clone is 1657 nt long, and encodes an ORF of 503 aa (Fig. 1). Within corresponding sequences, the clone is identical to the 550 nt long blpl cDNA ( Villand et al., 1992a), which was used as probe to screen leaf cDNA library in the present study. Based on nt sequence, blpl14 is the most homologous (95% identity) to a partial cDNA encoding the large subunit of AGPase from
wheat leaves (Olive et al., 1989). Identities of approx. 61–67% have been found between blpl14 and corresponding cDNAs for the large subunit of AGPase from other plants and/or tissues (e.g. Bhave et al., 1990; La Cognata et al., 1995), including barley seed endosperm ( Villand et al., 1992b). Based on aa sequence, the large subunit of barley leaf AGPase shares approx. 97 and 77% identity with the corresponding proteins from wheat (Olive et al., 1989) and potato (La Cognata et al., 1995) leaves, respectively, but only 36% identity with the AGPase from E. coli (Baecker et al., 1983). The AGPase shows little or no homology to a barley UDP-glucose pyrophosphorylase (approx. 22% identity, based on aa sequence), a related enzyme, which also utilizes glucose-1-P and PPi as substrates, but differs in its specificity for nucleoside triphosphates and sugar-nucleotides ( Eimert et al., 1996). The derived sequence contains three highly conserved Lys residues (Lys165, Lys456, Lys493) (Fig. 1), which were previously shown to be critical for binding of pyridoxal-P, an analog of AGPase activators, to the large subunit of spinach enzyme (Ball and Preiss, 1994). The two Lys residues located close to the C-terminus
Fig. 1. Nucleotide sequence of blpl14 and its derived aa sequence (GenBank accession No. U66876). Lys residues (Lys165, Lys456, Lys493) which are homologous to those previously shown for spinach AGPase to bind pyridoxal-P, an analog of activators of the enzyme (Ball and Preiss, 1994), are circled. A nt sequence consensus motif to the translation initiation site is underlined by thin line, and that characteristic of the termination signal for polymerase II (polyadenylation signal ) is underlined by thick line. A consensus motif for proteolytic processing of proteins that are targeted to plastid stroma (Bairoch, 1992) is boxed. An arrow denotes a putative transit peptide cleavage site.
K. Eimert et al. / Gene 189 (1997) 79–82
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are thought to lie near or at an allosteric site of the leaf enzyme. 2.3. Sequence motifs In the immediate vicinity of the first ATG codon at the 5∞ end of the cDNA clone, a sequence homology to the translation initiation region [(A/G)CCATGG ] ( Kozak, 1992) has been identified (Fig. 1). In this sequence, the positions of C bases are less conserved than those of other nt. In addition, in the 3∞-UTR region of blpl14, a motif characteristic of the termination signal for polymerase II (polyadenylation signal ) (AATAAA) (Proudfoot, 1991) was identified. When the derived aa sequence of blpl14 was analyzed for the possible presence of a transit peptide component, only one putative processing site was found. The sequence VAAA (aa 11–14), near the N-terminus of the derived protein (Fig. 1), matches a consensus motif for proteolytic processing of proteins that are post-translationally targeted to the plastid compartment. Based on the rules of Bairoch (1992), the cleavage site is (I/V-XA/C-3-A), where X denotes any aa. If the proposed processing site is indeed active in vivo, the processed barley protein would be expected to have a molecular mass of 53 562, which is close to the molecular mass of 54 kDa reported for the large subunit of purified barley leaf AGPase ( Kleczkowski et al., 1993). Because there is no in-frame stop codon upstream of the start codon at nt 41–43 of the cDNA, we are not certain if the complete transit peptide is encoded in the cDNA. It should be emphasized that throughout the whole aa sequence of Blpl14, there is no homology to a 19 aa long N-terminus sequence found for purified large subunit of AGPase from spinach leaves (Morell et al., 1987). This sequence is thought to be positioned immediately downstream of a transit peptide that is cleaved during post-translational processing (Morell et al., 1987). The lack of homology is not surprising, since the first 100 (or so) aa of the derived sequences of both subunit types of AGPase are known to be most divergent between plant species. In contrast to the leaf form of AGPase, both the small and large subunits of the endosperm isozyme do not have any recognizable transit peptide cleavage site in their derived aa sequences, which is consistent with the cytosolic location of the barley seed isozyme ( Villand and Kleczkowski, 1994; Thorbjørnsen et al., 1996a,b). 2.4. Expression in barley tissues The blpl14 cDNA was used as a molecular probe to study expression levels for the corresponding gene in barley tissues ( Fig. 2). The gene was found to be expressed preferentially in leaves rather than in seed endosperm, and there was no indication of any blpl14
Fig. 2. Expression patterns of genes encoding the large subunit of AGPase in barley tissues. (A) Northern blot of mRNAs from leaves (L), roots (R) and seed endosperm (E ) of barley probed with the blpl14 cDNA clone. (B) Northern blot of barley tissues probed with bepl10, a cDNA corresponding to the endosperm-specific form of the large subunit of AGPase ( Villand et al., 1992a,b). Methods: Poly-A+ RNA was extracted from 1 g quantities of barley leaves and roots, respectively, of 2–3-week-old greenhouse-grown plants (roots were obtained from plants grown on sand; leaves were from soil-grown plants), and from 400 mg barley endosperm (18 days after pollination), using mRNA isolation kit from Stratagene (La Jolla, CA, USA). Poly-A+ RNA (2 mg) was separated on a 1.2% agarose gel and blotted on nylon membrane according to the Northern technique (Sambrook et al., 1989). The hybridization was performed overnight at 65°C with 32P-labelled cDNA of blpl14 or bepl10, obtained by random priming using the Prime-a-Gene kit from Promega. For studies on the expression of blpl14 and bepl10 genes, the autoradiogram was exposed to the filter for 3 days and 1 day, respectively.
transcript in roots of barley. When the same blots were probed with bepl10, a homologous cDNA corresponding to the large subunit of AGPase that was isolated from a barley seed endosperm cDNA library ( Villand et al., 1992b), the hybridization was found only for the endosperm tissue (Fig. 2). A prolonged exposure of the autoradiogram (up to 10 days) did not bring about any signal for the leaf nor root transcripts (data not shown). We have previously detected a low level of the bepl10 transcript in roots of barley ( Villand et al., 1992a), but at that time roots were obtained from germinating seeds kept on moisted paper towels in the dark rather than from greenhouse-grown plants, as in the present study. Thus, it is possible that expression of bepl10 in roots may depend on growth conditions or developmental stage of this organ. The blpl14 and bepl10 share less than 63% identity, based on nt sequence, so they can be used as gene specific probes under experimental conditions used. On the other hand, the hybridization seen on Northern blots may also reflect the presence of some closely related yet unknown homologous transcript(s) for AGPase, especially with respect to the apparent expression of the blpl14 gene in seed endosperm (Fig. 2). Barley genome may contain at least four loci corresponding to the large subunit of AGPase ( Kilian et al., 1994). The differential expression of blpl14 and bepl10 transcripts in barley tissues is consistent with the view ( Kleczkowski et al., 1993; Villand and Kleczkowski,
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K. Eimert et al. / Gene 189 (1997) 79–82
1994; Thorbjørnsen et al., 1996b) that leaves and nonphotosynthetic tissues of this species contain distinct isozymes of AGPase, encoded by different transcripts.
Acknowledgement This research was supported, in part, by grants from the Swedish Natural Science Research Council and from the Swedish Foundation For Strategic Research.
References Baecker, P.A., Furlong, C.E. and Preiss, J. (1983) Primary structure of Escherichia coli ADP-glucose synthetase as deduced from the nucleotide sequence of the glgC gene. J. Biol. Chem. 258, 5084–5088. Bairoch, A. (1992) PC/GENE User and Reference Manual: Release 6.7, IntelliGenetics Inc. Ball, K. and Preiss, J. (1994) Allosteric sites of the large subunit of the spinach leaf ADP glucose pyrophosphorylase. J. Biol. Chem. 269, 24706–24711. Bhave, M.R., Lawrence, S., Barton, C. and Hannah, L.C. (1990) Identification and molecular characterization of shrunken-2 cDNA clones of maize. Plant Cell 2, 581–586. Eimert, K., Villand, P., Kilian, A. and Kleczkowski, L.A. (1996) Cloning and characterization of several cDNAs for UDP-glucose pyrophosphorylase from barley (Hordeum vulgare) tissues. Gene 170, 227–232. Kilian, A., Kleinhofs, A., Villand, P., Thorbjørnsen, T., Olsen, O.-A. and Kleczkowski, L.A. (1994) Mapping of ADP-glucose pyrophosphorylase genes from barley. Theor. Appl. Genet. 87, 869–871. Kleczkowski, L.A., Villand, P., Lo¨nneborg, A., Olsen, O.-A. and Lu¨thi, E. (1991) Plant ADP-glucose pyrophosphorylase: recent advances and biotechnological perspectives. Z. Naturforsch. 46c, 605–612. Kleczkowski, L.A., Villand, P., Preiss, J. and Olsen, O.-A. (1993) Kinetic mechanism and regulation of ADP-glucose pyrophosphory-
lase from barley (Hordeum vulgare) leaves. J. Biol. Chem. 268, 6228–6233. Kozak, M. (1992) Regulation of translation in eukaryotic systems. Annu. Rev. Cell Biol. 8, 197–225. La Cognata, U., Willmitzer, L. and Mu¨ller-Ro¨ber, B. (1995) Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase. Mol. Gen. Genet. 246, 538–548. Morell, M.K., Bloom, M., Knowles, V. and Preiss, J. (1987) Subunit structure of spinach leaf ADP-glucose pyrophosphorylase. Plant Physiol. 85, 182–187. Nelson, O. and Pan, D. (1995) Starch synthesis in maize endosperm. Annu. Rev. Plant. Physiol. Plant Mol. Biol. 46, 475–496. Olive, M.R., Ellis, R.J. and Schuch, W.W. (1989) Isolation and nucleotide sequences of cDNA clones encoding ADP-glucose pyrophosphorylase polypeptides from wheat leaf and endosperm. Plant Mol. Biol. 12, 525–538. Proudfoot, N.J. (1991) Poly(A) signals. Cell 64, 671–674. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Thorbjørnsen, T., Villand, P., Denyer, K., Olsen, O.-A. and Smith, A.M. (1996a) Distinct isoforms of ADP-glucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm. Plant J. 10, 243–250. Thorbjørnsen, T., Villand, P., Kleczkowski, L.A. and Olsen, O.A. (1996b) A single gene encodes two different transcripts for the ADPglucose pyrophosphorylase small subunit from barley (Hordeum vulgare). Biochem. J. 313, 149–154. Villand, P. and Kleczkowski, L.A. (1994) Is there an alternative pathway for starch biosynthesis in cereal seeds? Z. Naturforsch. 49c, 215–219. Villand, P., Aalen, R., Olsen, O.-A., Lu¨thi, E., Lo¨nneborg, A. and Kleczkowski, L.A. (1992a) PCR-amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues. Plant Mol. Biol. 19, 381–389. Villand, P., Olsen, O.-A., Kilian, A. and Kleczkowski, L.A. (1992b) ADP-glucose pyrophosphorylase large subunit cDNA from barley endosperm. Plant Physiol. 100, 1617–1618. Villand, P., Olsen, O.-A. and Kleczkowski L.A. (1993) Molecular characterization of multiple cDNA clones of ADP-glucose pyrophosphorylase from Arabidopsis thaliana. Plant Mol. Biol. 23, 1279–1284.
Molecular cloning and expression of the large subunit of ADP-glucose pyrophosphorylase from barley (Hordeum vulgare) leaves Klaus Eimert a,1, Cheng Luo a, Annabelle De´jardin a, Per Villand a,b, Tine Thorbjørnsen b,2, Leszek A. Kleczkowski a,* a Department of Plant Physiology, Umea˚ University, 901-87 Umea˚, Sweden b Plant Molecular Biology Laboratory, Agricultural University of Norway, 1432 Aas, Norway Received 30 August 1996; revised 28 October 1996; accepted 29 October 1996; Received by J. Wild
Abstract A cDNA clone, blpl14, corresponding to the large subunit of ADP-glucose pyrophosphorylase (AGPase), has been isolated from a cDNA library prepared from leaves of barley (Hordeum vulgare L.). An open reading frame encodes a protein of 503 aa, with a calculated molecular weight of 54 815. The derived aa sequence contains a putative transit peptide sequence, required for targeting to plastids, and has a highly conserved positioning of critical Lys residues that are believed to be involved in effector binding. The derived aa sequence shows 97% identity with the corresponding protein from wheat, but only 36% identity with AGPase from E. coli. The blpl14 gene is expressed predominantly in leaves and to a lesser degree in seed endosperm, but not roots, of barley. Keywords: Chloroplast; Endosperm; Starch; Sucrose; Transit peptide
1. Introduction ADP-glucose pyrophosphorylase (AGPase) is the key enzyme of starch biosynthesis in all plants and bacteria. In plants, the enzyme is composed of two subunit types, encoded by different genes (Morell et al., 1987; Kleczkowski et al., 1991; Nelson and Pan, 1995). In barley, at least two isozymes of AGPase have been described, one in seed endosperm and the other – in leaves ( Kleczkowski et al., 1993). The isozymes differ in their kinetic properties and regulation ( Kleczkowski et al., 1993), and are localized in different cell compartments ( Villand and Kleczkowski, 1994; Thorbjørnsen et al., 1996a,b). Although several cDNA sequences for plant AGPase * Corresponding author. Tel. +46 90 167781; Fax +46 90 166676; e-mail: [email protected] 1 Current address: Department of Botany, Geisenheim Research Centre, 65366 Geisenheim, Germany. 2 Current address: Plant Biochemistry Laboratory, Royal Veterinary and Agricultural University, Thorvaldsens vej 40, Copenhagen, Denmark. Abbreviations: aa, amino acid(s); AGPase, ADP-glucose pyrophosphorylase; nt, nucleotide(s); ORF, open reading frame; UTR, untranslated region(s). 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 8 37 - 2
have already been published (summarized in La Cognata et al., 1995), there are only few reports describing isolation of cDNA(s) for the large subunit of the leaf form of the enzyme (Olive et al., 1989; Villand et al., 1992a, 1993; La Cognata et al., 1995). Previously, we have used a PCR technique to amplify several partial (approx. 550 nt) cDNAs corresponding to small and large subunits of AGPase from various plant tissues ( Villand et al., 1992a, 1993), including leaves. These PCR products have proven to be very useful as genespecific probes to isolate full length cDNAs, genomic clones and for expression studies in barley ( Villand et al., 1992b; Thorbjørnsen et al., 1996b). In the present study, isolation of a cDNA encoding ORF of the leaf form of the large subunit of barley AGPase is reported, along with characterization of its expression in barley tissues.
2. Experimental and discussion 2.1. Cloning of blpl14 The isolation of cDNA encoding AGPase was carried out by screening of a cDNA library prepared from
80
K. Eimert et al. / Gene 189 (1997) 79–82
mRNA isolated from leaves (3 weeks old ) of barley (Hordeum vulgare L. cv. Bomi). The library was custommade by Clontech (Palo Alto, CA, USA) using the l ZAPII system. A 550-nt blpl cDNA clone, previously isolated by PCR amplification on total barley leaf mRNA ( Villand et al., 1992a), was used as the hybridization probe. A putative full-length clone, blpl14, was sequenced on both strands using 32P-end-labelled custom-made primers (fmol DNA Sequencing, Promega, Madison, WI, USA).
2.2. Nucleotide and deduced amino-acid sequences A cDNA clone, blpl14 (GenBank accession No. U66876), encoding the large subunit of AGPase has been isolated from a barley leaf cDNA library. The clone is 1657 nt long, and encodes an ORF of 503 aa (Fig. 1). Within corresponding sequences, the clone is identical to the 550 nt long blpl cDNA ( Villand et al., 1992a), which was used as probe to screen leaf cDNA library in the present study. Based on nt sequence, blpl14 is the most homologous (95% identity) to a partial cDNA encoding the large subunit of AGPase from
wheat leaves (Olive et al., 1989). Identities of approx. 61–67% have been found between blpl14 and corresponding cDNAs for the large subunit of AGPase from other plants and/or tissues (e.g. Bhave et al., 1990; La Cognata et al., 1995), including barley seed endosperm ( Villand et al., 1992b). Based on aa sequence, the large subunit of barley leaf AGPase shares approx. 97 and 77% identity with the corresponding proteins from wheat (Olive et al., 1989) and potato (La Cognata et al., 1995) leaves, respectively, but only 36% identity with the AGPase from E. coli (Baecker et al., 1983). The AGPase shows little or no homology to a barley UDP-glucose pyrophosphorylase (approx. 22% identity, based on aa sequence), a related enzyme, which also utilizes glucose-1-P and PPi as substrates, but differs in its specificity for nucleoside triphosphates and sugar-nucleotides ( Eimert et al., 1996). The derived sequence contains three highly conserved Lys residues (Lys165, Lys456, Lys493) (Fig. 1), which were previously shown to be critical for binding of pyridoxal-P, an analog of AGPase activators, to the large subunit of spinach enzyme (Ball and Preiss, 1994). The two Lys residues located close to the C-terminus
Fig. 1. Nucleotide sequence of blpl14 and its derived aa sequence (GenBank accession No. U66876). Lys residues (Lys165, Lys456, Lys493) which are homologous to those previously shown for spinach AGPase to bind pyridoxal-P, an analog of activators of the enzyme (Ball and Preiss, 1994), are circled. A nt sequence consensus motif to the translation initiation site is underlined by thin line, and that characteristic of the termination signal for polymerase II (polyadenylation signal ) is underlined by thick line. A consensus motif for proteolytic processing of proteins that are targeted to plastid stroma (Bairoch, 1992) is boxed. An arrow denotes a putative transit peptide cleavage site.
K. Eimert et al. / Gene 189 (1997) 79–82
81
are thought to lie near or at an allosteric site of the leaf enzyme. 2.3. Sequence motifs In the immediate vicinity of the first ATG codon at the 5∞ end of the cDNA clone, a sequence homology to the translation initiation region [(A/G)CCATGG ] ( Kozak, 1992) has been identified (Fig. 1). In this sequence, the positions of C bases are less conserved than those of other nt. In addition, in the 3∞-UTR region of blpl14, a motif characteristic of the termination signal for polymerase II (polyadenylation signal ) (AATAAA) (Proudfoot, 1991) was identified. When the derived aa sequence of blpl14 was analyzed for the possible presence of a transit peptide component, only one putative processing site was found. The sequence VAAA (aa 11–14), near the N-terminus of the derived protein (Fig. 1), matches a consensus motif for proteolytic processing of proteins that are post-translationally targeted to the plastid compartment. Based on the rules of Bairoch (1992), the cleavage site is (I/V-XA/C-3-A), where X denotes any aa. If the proposed processing site is indeed active in vivo, the processed barley protein would be expected to have a molecular mass of 53 562, which is close to the molecular mass of 54 kDa reported for the large subunit of purified barley leaf AGPase ( Kleczkowski et al., 1993). Because there is no in-frame stop codon upstream of the start codon at nt 41–43 of the cDNA, we are not certain if the complete transit peptide is encoded in the cDNA. It should be emphasized that throughout the whole aa sequence of Blpl14, there is no homology to a 19 aa long N-terminus sequence found for purified large subunit of AGPase from spinach leaves (Morell et al., 1987). This sequence is thought to be positioned immediately downstream of a transit peptide that is cleaved during post-translational processing (Morell et al., 1987). The lack of homology is not surprising, since the first 100 (or so) aa of the derived sequences of both subunit types of AGPase are known to be most divergent between plant species. In contrast to the leaf form of AGPase, both the small and large subunits of the endosperm isozyme do not have any recognizable transit peptide cleavage site in their derived aa sequences, which is consistent with the cytosolic location of the barley seed isozyme ( Villand and Kleczkowski, 1994; Thorbjørnsen et al., 1996a,b). 2.4. Expression in barley tissues The blpl14 cDNA was used as a molecular probe to study expression levels for the corresponding gene in barley tissues ( Fig. 2). The gene was found to be expressed preferentially in leaves rather than in seed endosperm, and there was no indication of any blpl14
Fig. 2. Expression patterns of genes encoding the large subunit of AGPase in barley tissues. (A) Northern blot of mRNAs from leaves (L), roots (R) and seed endosperm (E ) of barley probed with the blpl14 cDNA clone. (B) Northern blot of barley tissues probed with bepl10, a cDNA corresponding to the endosperm-specific form of the large subunit of AGPase ( Villand et al., 1992a,b). Methods: Poly-A+ RNA was extracted from 1 g quantities of barley leaves and roots, respectively, of 2–3-week-old greenhouse-grown plants (roots were obtained from plants grown on sand; leaves were from soil-grown plants), and from 400 mg barley endosperm (18 days after pollination), using mRNA isolation kit from Stratagene (La Jolla, CA, USA). Poly-A+ RNA (2 mg) was separated on a 1.2% agarose gel and blotted on nylon membrane according to the Northern technique (Sambrook et al., 1989). The hybridization was performed overnight at 65°C with 32P-labelled cDNA of blpl14 or bepl10, obtained by random priming using the Prime-a-Gene kit from Promega. For studies on the expression of blpl14 and bepl10 genes, the autoradiogram was exposed to the filter for 3 days and 1 day, respectively.
transcript in roots of barley. When the same blots were probed with bepl10, a homologous cDNA corresponding to the large subunit of AGPase that was isolated from a barley seed endosperm cDNA library ( Villand et al., 1992b), the hybridization was found only for the endosperm tissue (Fig. 2). A prolonged exposure of the autoradiogram (up to 10 days) did not bring about any signal for the leaf nor root transcripts (data not shown). We have previously detected a low level of the bepl10 transcript in roots of barley ( Villand et al., 1992a), but at that time roots were obtained from germinating seeds kept on moisted paper towels in the dark rather than from greenhouse-grown plants, as in the present study. Thus, it is possible that expression of bepl10 in roots may depend on growth conditions or developmental stage of this organ. The blpl14 and bepl10 share less than 63% identity, based on nt sequence, so they can be used as gene specific probes under experimental conditions used. On the other hand, the hybridization seen on Northern blots may also reflect the presence of some closely related yet unknown homologous transcript(s) for AGPase, especially with respect to the apparent expression of the blpl14 gene in seed endosperm (Fig. 2). Barley genome may contain at least four loci corresponding to the large subunit of AGPase ( Kilian et al., 1994). The differential expression of blpl14 and bepl10 transcripts in barley tissues is consistent with the view ( Kleczkowski et al., 1993; Villand and Kleczkowski,
82
K. Eimert et al. / Gene 189 (1997) 79–82
1994; Thorbjørnsen et al., 1996b) that leaves and nonphotosynthetic tissues of this species contain distinct isozymes of AGPase, encoded by different transcripts.
Acknowledgement This research was supported, in part, by grants from the Swedish Natural Science Research Council and from the Swedish Foundation For Strategic Research.
References Baecker, P.A., Furlong, C.E. and Preiss, J. (1983) Primary structure of Escherichia coli ADP-glucose synthetase as deduced from the nucleotide sequence of the glgC gene. J. Biol. Chem. 258, 5084–5088. Bairoch, A. (1992) PC/GENE User and Reference Manual: Release 6.7, IntelliGenetics Inc. Ball, K. and Preiss, J. (1994) Allosteric sites of the large subunit of the spinach leaf ADP glucose pyrophosphorylase. J. Biol. Chem. 269, 24706–24711. Bhave, M.R., Lawrence, S., Barton, C. and Hannah, L.C. (1990) Identification and molecular characterization of shrunken-2 cDNA clones of maize. Plant Cell 2, 581–586. Eimert, K., Villand, P., Kilian, A. and Kleczkowski, L.A. (1996) Cloning and characterization of several cDNAs for UDP-glucose pyrophosphorylase from barley (Hordeum vulgare) tissues. Gene 170, 227–232. Kilian, A., Kleinhofs, A., Villand, P., Thorbjørnsen, T., Olsen, O.-A. and Kleczkowski, L.A. (1994) Mapping of ADP-glucose pyrophosphorylase genes from barley. Theor. Appl. Genet. 87, 869–871. Kleczkowski, L.A., Villand, P., Lo¨nneborg, A., Olsen, O.-A. and Lu¨thi, E. (1991) Plant ADP-glucose pyrophosphorylase: recent advances and biotechnological perspectives. Z. Naturforsch. 46c, 605–612. Kleczkowski, L.A., Villand, P., Preiss, J. and Olsen, O.-A. (1993) Kinetic mechanism and regulation of ADP-glucose pyrophosphory-
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