Plant Cell Reports
Plant Cell Reports (1994) 14:180-183
,~ Springer-Verlag 1994
Cloning and expression analysis of an ADP-ribosylation factor from Solanum tuberosum L. Jan S z o p a t and Bernd Miiller-Riiber 2 I Institute o f Biochemistry, University o f Wroclaw, Przybyszewskiego 63, 51-148 Wroclaw, Poland 2 Institut fiir Genbiologische F o r s c h u n g Berlin G m b H , lhnestralSe 63, D-14195 Berlin, G e r m a n y Received 3 February 1994/Revised version received 20 April 1994 - C o m m u n i c a t e d by H. L6rz
Summary. A cDNA clone encoding an ADP-ribosylation factor from potato (Solanum tuberosum L.) was isolated. The nucleotide and deduced amino acid sequences show high homology to known ADP-ribosylation factor sequences from Arabidopsis, yeast, cow and man. In northern blot experiments, all tissues analysed showed expression of the corresponding mRNA. Strongest expression was found, however, in potato tubers.
ARF, ADP-ribosylation factor; BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulfate; SSC, sodium saline citrate.
Abbreviations:
Introduction
ADP-ribosylation factor (ARF) proteins, a group of small GTP-binding proteins of ca. 21 kDa Mr, are required in coated vesicle assembly in the Golgi apparatus (Serafini et al. 1991; Orci et al. 1993). ARF was originally identified in mammalian cells as a protein cofactor required for efficient in vitro ADP-ribosylation of the e~ subunit of the trimeric G protein Gs, thereby leading to activation of adenylate cyclase (Cassel and Pfeuffer 1978; Schleifer et al. 1982; Kahn and Gilman 1986). ARFs are highly conserved proteins in eukaryotic cells. Immunological data showed that ARF is concentrated in the Golgi apparatus (Steams et al. 1990b). In recent years, a large number of ARF and ARF-like cDNA and genomic clones have been obtained from the fungi, animals and the plant Arabidopsis thaliana. Two ARF genes (ARF1 and ARF2) exist in S. cerevisiae (Stearns et al. 1990a). Disruption of ARF1 (encoding the major ARF protein in yeast) results in slow growth and cold sensitivity. No discernible phenotype occurred upon loss of ARF2, whereas double ARF disruption was lethal Correspondence to: J. Szopa
(Steams et al. 1990a). At least six different ARFs, grouped into three classes, have been identified in mammals (Moss and Vaughan 1993). ARF proteins are ubiquitously expressed in mammalian tissues, but are in addition subject to developmental and hormonal regulation (Duman et al. 1990; Tsai et al. 1991). Recently, the isolation of a cDNA encoding ARF from Arabidopsis thaliana was reported (Regad et al. 1993). A single 0.9 kb transcript was detected in RNA isolated from cell suspension cultures. The expression of the ARF mRNA was nearly constant throughout the different stages of suspension growth (Regad et al. 1993). Although only one transcript was found, at least two distinct genomic clones have been isolated from an Arabidopsis genomic library (Regad et al. 1993), indicating a complex situation also in higher plants. Here we report the cloning and expression analysis of a cDNA encoding an ARF from potato (Solanum tuberosum L.). The ARF cDNA described here was isolated due to a partial sequence homology with a 14-3-3 protein cDNA probe from Cucurbita (Szopa, EMBL/Genbank accession number X76086).
Materials and methods Plant material Potato plants (Solanura tuberosum L. cv. Desiree) were obtained through Saatzucht Fritz Lange KG (Bad Schwartau, Germany).
Plants were cultivated in soft in a temperature regulated greenhouse under a light/dark regime of approximately 16 h light (22~ and 8 h dark (15~ Plants were grown in individual pots (area 200 cm2, 15 cm deep) and were watered daily. Recombinant DNA techniques. DNA manipulations were done essentially as described in Sambrook et al. (1989). DNA restriction and modification enzymes were obtained from Boehringer Mannheim (Germany) and New England Biolabs (Beverly, MA). Escherichia coli strains DHSo. and XL1Blue were used for bacterial work, cDNA library screening. A potato leaf cDNA library prepared in R. ZAP II (Kossmann et at. 1992) was used for screening. A 1200 bp long Xbal/XhoI
181
cross-hybridizing phages were plaque-purified and converted to pBluescript derivatives by in vivo excision. Partial DNA sequences were obtained for the cDNA inserts of isolated phagemid clones. Several cDNA clones encoding 14-3-3 proteins from potato were identified (Szopa, unpublished). Besides these, one of the cDNA clones, called PA215, encoded a protein showing high homology to ARF proteins from other organisms. The entire nucleotide sequence of the potato ARF cDNA was obtained and found to display partial homology to the Cucurbita cDNA that was used as a screening probe (see Discussion). The potato clone contained a 637 bp long insert including an open reading frame of 591 nucleotides encoding a protein of 197 amino acids. The calculated molecular mass is 22.6 kDa. A rather short untranslated sequence was present at the 3" end of the cDNA and no poly(A)-tail was present (which, however, was frequently observed in the cDNA library used for screening and therefore does not appear to be a special feature of the ARF cDNA; Muller-Rober, unpublished observations). The Solanum tuberosum ARF cDNA sequence has been deposited in the EMBL/GenBank database under accession number X74461. A comparison between the potato ARF amino acid sequence with ARF proteins from other species is shown in Fig. 1. The sequences are highly conserved. When the potato ARF sequence was compared on the nucleotide level with other known sequences in the GenEMBL database, highest homology (78.4% identity in a 587 bp overlap) was found to the Arabidopsis thaliana ARF sequence. Very high homology was also found to several ARF sequences from cow (up to 79% identity in a 542 bp overlap), fungi (e.g. Candida albicans ARF1; 72.6% identity in 572 bp overlap) and man (76.1% identity in 549 bp overlap). Comparison of the deduced potato ARF amino acid sequence with sequences in the SwissProt databank revealed highest homology to the ARF1 protein from man (92.3% identity in 181 amino acid overlap). A lower degree
cDNA insert of plasmid a215 encoding a 14-3-3 protein (Ichimura et al. 1988) of Cucurbita (EMBL/GenBank accession number X76086) was used as a hybridization probe. Radioactive labelling was performed using a multiprime labelling kit (Boehringer Mannheim). Hybridization was done overnight at 42~ in 250 ram sodium phosphate buffer (pH 7.2) containing 7% (w/v) SDS, 1% (w/v) BSA and 1 mM EDTA. Filters were washed in 0.1 x SSC, 0.5% (w/v) SDS at 42 ~ C for 30 rnin. Plaque-purified phages were subjected to in vivo excision according to the manufacturer's (Stratagene) conditions. Rescued phagemid clones were investigated by restriction analysis and DNA sequencing. DNA sequencing and data analysis o.f potato ARF cDNA. DNA sequences were determined by the dideoxy method (Sanger et al. 1977). Commercial sequencing primers (Pharmacia, Freiburg, Germany) and specifically synthesized oligonucleotides (TibMolbiol, Berlin, Germany) were used for plasmid sequencing with T7 DNA polymerase (pharmacia). Sequence analysis was performed using the programs of the University of Wisconsin Genetics Computer Group (GCG Package, Version 7) (Devereux et al. 1984). Sequence comparisons on cDNA and deduced amino acid sequences were performed in GenBank, EMBL and SwissProt databases using the FASTA search program (Pearson and Lipman 1988). Northern blot analysis. Total RNA was prepared from frozen plant material using the guanidinium hydrochloride method as described by Logemann et al. (1987). Following electrophoresis (1.5% [w/v] agarose, 15% [v/v] formaldehyde) RNA w ~ transferred to nylon membranes (Hybond N, Amersham, UK). Membranes were hybridized overnight at 65~ in the buffer described in section describing cDNA library screening. The EcoRI cDNA insert of the plasmid PA215 (see Results) was used as a radioactively labelled hybridization probe. Filters were washed three times in 0.1 x SSC, 0.5% (w/v) SDS for 30 rain at 65~
Results Isolation and sequence analysis of a potato ARF cDNA
A potato leaf cDNA library was screened with a Cucurbita 14-3-3 protein cDNA probe under moderately stringent conditions as described in Materials and me~ods. Several A t a r f . Pep Boarf. Pep Hsa~f.Pep Scarf. Pep SCarf.Pep At:arf .Pep Boaxf.Pep H~axf.Pep Scaxf.Pep S~.arf.Pep A t a r f . Pep Soa~f.Pep llsar f .Pep Scarf.Pep Sl:arf .Pep
Atarf.Pep
Boarf.Pep Hsarf.Pep Scarf.Pep
Staff.Pep
I~A~Lr~alLF0~KZ
RIL
TISIKLPS]EL~L_.~AIKKEHR~
VG
DAA~KTT
VG
D.AAGKTTILTKLKL
LT~LK~
GGEEZVTTI ~ T EVITTI ~ GE V T T r P TTI
C
SO 50 50
GQDKIRPLWRHYTQHTQGLIPVVDSNDRDRV~ F~VETVETKNI FTVWDVGGQDKIRPLWRHYPQNTQGLIFV~DSNDASRV[ FNV~TV,TKNIS,TVWDVGGQDKIRPL,~HTFONT~GLIFV DS~DXERV[ , . v , v [ ] T x . Sr,vwDvc~QD@,~[]Lw S , , [ ] N , m C ~ , r V ~ D S D~RII FNVETVETKNI.SFTVWDVG ~DKIRPLWRHTFQNT~LIFV DSHDRORV|
I00
HEARS
ISO
I~HVETVSYKMI~FTVMDV g LSRMLAEDELXDAVLLVFANKQD
pgAM.AAEI
~sx~E~n~xxL~sss~x~xf'X-~vrx~x~P~x~xx~s , r. s ~ T x s ~ LSNQL~QK] NJWTIQAT T~GD EGLD NIWTIQATCAT G D G L T E G L D WL S N Q L R 14QKI PIWPIQATCATSGEGLTSGLE ,. s ~ t ~ HIWYTQ~TCATSGEGLTEGLD
LG
TD
~
HSLXIH
us~
I00
100
loo 100
~s0 lal
tSt
181 9
181
Fig. 1. Alignment of the deduced ARF amino acid sequence from Solanum tuberosum L and other species. The sequences are described in: Atarf - Arabidapsis thaliana (Regad et al. 1993); Boarf - cow (Sewell and Kahn 1988); Hsarf - man (Kahn et al. 1991); Scarf - Saccharomyces cerevisiae (Sewell and Kahn 1988); Staff -Solanum tuberosum (this report). Numbers indicate amino acid positions.
182 of homology was found to bovine ARF and several other human ARFs, as well as to ARFs from Saccharomyces cerevisiae (79.4% identity to ARF2 and 78.8% identity to ARF1), Giardia lamblia (68.6%) and chicken (67.4%). Appreciable homology to ARF-like proteins (Tamkun et al. 1991) of Drosophila melanogaster (56.6% identity) was also identified. Several conserved motifs, which are present in ARF proteins, are also observed in the potato ARF sequence shown here. Three consensus sequences proposed to be involved in GTP binding (Kaziro et al. 1991) are present at amino acid positions 24 - 31 (sequence GLDAAGKT; P motif of ARF proteins), 67 - 71 (sequence DVGGQ, G" motif DXXGQ), and 126 - 129 (sequence NKQD, G motif NKXD). ARF proteins usually have a length of 180 or 181 amino acids. Interestingly, potato ARF contains a Cterminal extension (16 amino acids long) which is not present in most of the other ARFs. The role of this extension is not known.
Expression analysis of potato ARF In order to investigate the mRNA expression of ARF, northern blot experiments were performed. Total RNA was isolated from various tissues of a collection of 4 independent 10 - 12 weeks old potato plants and after electrophoretic separation hybridized against the potato ARF cDNA probe. In all tissues tested, a ca. 0.9 kb long transcript was detected under high stringency conditions (Fig. 2). The strongest expression was found in growing tubers of different developmental stages, although the expression level varied between individual tubers. A strong expression was also seen in roots, whereas in sink and source leaves of the potato plants usually a lower level of mRNA was detected.
Discussion By screening a potato leaf cDNA library, we have identified a cDNA encoding a protein with high homology to ADP-ribosylation factor (ARF) proteins from a variety of other organisms, including animals and fungi. The ARF cDNA was identified in a heterologous screening procedure using a cucurbita eDNA encoding a 14-3-3 protein (Ichimura et al. 1988). When the deduced amino acid sequence of the potato ARF (see Fig. 1) was compared with the deduced amino acid sequence of the Cucurbita 14-3-3 protein no significant homology between the two proteins was found (not shown). However, a 49 bp long region of high homology (84%) was detected between the two cDNAs (not shown) which could explain the crosshybridization during the screening. Although ARF and 14-3-3 proteins do not have the same function, the recently published observation that the bovine FAS protein (a member of the 14-3-3 protein family) is able to activate
Fig. 2. Northernblot analysisof ARF gene expressionin varioustissuesof potato plants. Total RNA (501.tg/lane) was fractionated by denaturing agarosegel electrophoresisand aftertransferto nylonmembraneshybridized againstthe 32p-labelledpotatoARFeDNAisolatedfromclonePA215.T1, T2, T3: developingtubers of differentdevelopmentalstages; R: root; G: gynoeceum;St: stolon;So: sourceleaf;Si: sinkleaf. ADP-ribosylation (Fu et al. 1993) is interesting. The mRNA corresponding to the ARF cDNA described here is expressed in all tissues analysed. In addition, several experimental treatments, like incubation of detached leaves in light or darkness, or feeding of sucrose or nitrate to the leaves, did not significantly influence ARF mRNA levels (data not shown). Likewise, in Arabidopsis it has been shown that ARF mRNA expression during the growth cycle of a cell culture, including the stationary phase, was nearly constant. The function of ARF proteins in higher plants is not known at present. Since ARF expression is rather high in the various potato tissues and not strongly influenced by various environmental conditions (at least not in short term experiments, i.e. up to 48 h of treatment) it might be concluded that plant ARFs play a rather general (or housekeeping) function. No mutants deficient in ARF proteins have been described for higher plants. Loss of functional ARF proteins could be lethal to the plant, or the absence of ARF proteins is not accompanied by an easily visible phenotype. The cloning of the potato ARF eDNA described in this report will allow an alternative approach to investigate the role of ARF proteins: the cDNA can be used to down-regulate the levels of ARF mRNA (and thereby corresponding ARF protein) via the antisense RNA technique in transgenic plants (Van der Krol et al. 1988). Usually, antisense inhibition varies between independent transformants. Therefore, even in the case that complete deficiency in ARF proteins is lethal, it should be possible to recover transgenic plants which have only partially lost the
183 ARF protein. Preliminary data show that ARF mRNA levels can strongly be down-regulated in transgenic potato plants without severe changes in plant development (Szopa and Muller-Rober, unpublished data). The antisense plants hopefully will allow to assign a function to plant ARF proteins. Acknowledgement. This work was supported by Grant 400 88 91 01 from KI3N, and by the DFG.
References
Cassel D and Pfeuffer T (1978) Proc Natl Acad Sci USA 75:2669-2673 Devereux J, Haeberli P and Smithies O (1984) Nucl Acids Res 12:387-395 Duman RS, Winston SM, Clark JA and Nestler EJ (1990) J Neurochem 55:1813-1816 Fu H, Cobum J and Collier RJ (1993) Proc Natl Acad Sci USA 90:2320-2324 Ichimura T, Isobe T, Okuyama T, Takahashi N, Kazuaki A, Kuwano R and Takahashi Y (1988) Proc Natl Acad Sci USA 85:7084-7088 Kahn RA and Gilman AG (1986) J Biol Chem 261: 79067911 Kahn RA, Kern FG, Clark J, Gelmann EP and Rulka C (1991) J Biol Chem 266:2606-2614 Kaziro Y, Itoh H, Kozasa T, Nakafuko M and Satoh T (1991) Annu Rev Biochem 60:349-400 Kossmann J, Muller-Rober B, Dyer TA, Raines CA, Sonnewald U and Willmitzer L (1992) Planta 188: 712 Logemann J, Schell J and Willmitzer L (1987) Anal Biochem 163:21-26 Moss J and Vaughan M (1993) Cellular Signalling 5: 367379 Orci L, Palmer DJ, Amherdt M and Rothman JE (1993) Nature 364:732-734 Pearson WR and Lipman DJ (1988) Proc Natl Acad Sci USA 85:2444-2448 Regad F, Bardet C, Tremousaygue D, Moisan A, Lescure B and Axelos M (1993) FEBS Lett 316:133-136 Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press Sanger F, Nicklen S and Coulson AR (1977) Proc Natl Acad Sci USA 74:5463-5467 Schleifer LS, Kahn RA, Hanski E, Northup JK, Sternweis PC and Gilman AG (1982) J Biol Chem 257:20-23 Serafini T, Orci L, Amherdt M, Brunner M, Kahn RA and Rothman JE (1991) Cell 67:239-253 Sewell JL and Kahn RA (1988) Proc Natl Acad Sci USA 85:4620-4624 Stearns T, Kahn RA, Botstein D and Hoyt MA (1990a) Mol Cell Biol 10:6690-6699
Stearns T, Willingham MC, Botstein D and Kahn RA (1990b) Proc Natl Acad Sci USA 87:1238-1242 Tamkun JW, Kahn RA, Kissinger M, Brizuela B J, Rulka C, Scott MP and Kennison JA (1991) Proc Natl Acad Sci USA 88:3120-3124 Tsai S-C, Adamik R, Tsuchiya M, Chang PP, Moss J and Vaughan M (1991) J Biol Chem 266:8213-8219 Van der Krol A, Mol J and Stuitje A (1988) BioTechniques 6:958-976
Plant Cell Reports (1994) 14:180-183
,~ Springer-Verlag 1994
Cloning and expression analysis of an ADP-ribosylation factor from Solanum tuberosum L. Jan S z o p a t and Bernd Miiller-Riiber 2 I Institute o f Biochemistry, University o f Wroclaw, Przybyszewskiego 63, 51-148 Wroclaw, Poland 2 Institut fiir Genbiologische F o r s c h u n g Berlin G m b H , lhnestralSe 63, D-14195 Berlin, G e r m a n y Received 3 February 1994/Revised version received 20 April 1994 - C o m m u n i c a t e d by H. L6rz
Summary. A cDNA clone encoding an ADP-ribosylation factor from potato (Solanum tuberosum L.) was isolated. The nucleotide and deduced amino acid sequences show high homology to known ADP-ribosylation factor sequences from Arabidopsis, yeast, cow and man. In northern blot experiments, all tissues analysed showed expression of the corresponding mRNA. Strongest expression was found, however, in potato tubers.
ARF, ADP-ribosylation factor; BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulfate; SSC, sodium saline citrate.
Abbreviations:
Introduction
ADP-ribosylation factor (ARF) proteins, a group of small GTP-binding proteins of ca. 21 kDa Mr, are required in coated vesicle assembly in the Golgi apparatus (Serafini et al. 1991; Orci et al. 1993). ARF was originally identified in mammalian cells as a protein cofactor required for efficient in vitro ADP-ribosylation of the e~ subunit of the trimeric G protein Gs, thereby leading to activation of adenylate cyclase (Cassel and Pfeuffer 1978; Schleifer et al. 1982; Kahn and Gilman 1986). ARFs are highly conserved proteins in eukaryotic cells. Immunological data showed that ARF is concentrated in the Golgi apparatus (Steams et al. 1990b). In recent years, a large number of ARF and ARF-like cDNA and genomic clones have been obtained from the fungi, animals and the plant Arabidopsis thaliana. Two ARF genes (ARF1 and ARF2) exist in S. cerevisiae (Stearns et al. 1990a). Disruption of ARF1 (encoding the major ARF protein in yeast) results in slow growth and cold sensitivity. No discernible phenotype occurred upon loss of ARF2, whereas double ARF disruption was lethal Correspondence to: J. Szopa
(Steams et al. 1990a). At least six different ARFs, grouped into three classes, have been identified in mammals (Moss and Vaughan 1993). ARF proteins are ubiquitously expressed in mammalian tissues, but are in addition subject to developmental and hormonal regulation (Duman et al. 1990; Tsai et al. 1991). Recently, the isolation of a cDNA encoding ARF from Arabidopsis thaliana was reported (Regad et al. 1993). A single 0.9 kb transcript was detected in RNA isolated from cell suspension cultures. The expression of the ARF mRNA was nearly constant throughout the different stages of suspension growth (Regad et al. 1993). Although only one transcript was found, at least two distinct genomic clones have been isolated from an Arabidopsis genomic library (Regad et al. 1993), indicating a complex situation also in higher plants. Here we report the cloning and expression analysis of a cDNA encoding an ARF from potato (Solanum tuberosum L.). The ARF cDNA described here was isolated due to a partial sequence homology with a 14-3-3 protein cDNA probe from Cucurbita (Szopa, EMBL/Genbank accession number X76086).
Materials and methods Plant material Potato plants (Solanura tuberosum L. cv. Desiree) were obtained through Saatzucht Fritz Lange KG (Bad Schwartau, Germany).
Plants were cultivated in soft in a temperature regulated greenhouse under a light/dark regime of approximately 16 h light (22~ and 8 h dark (15~ Plants were grown in individual pots (area 200 cm2, 15 cm deep) and were watered daily. Recombinant DNA techniques. DNA manipulations were done essentially as described in Sambrook et al. (1989). DNA restriction and modification enzymes were obtained from Boehringer Mannheim (Germany) and New England Biolabs (Beverly, MA). Escherichia coli strains DHSo. and XL1Blue were used for bacterial work, cDNA library screening. A potato leaf cDNA library prepared in R. ZAP II (Kossmann et at. 1992) was used for screening. A 1200 bp long Xbal/XhoI
181
cross-hybridizing phages were plaque-purified and converted to pBluescript derivatives by in vivo excision. Partial DNA sequences were obtained for the cDNA inserts of isolated phagemid clones. Several cDNA clones encoding 14-3-3 proteins from potato were identified (Szopa, unpublished). Besides these, one of the cDNA clones, called PA215, encoded a protein showing high homology to ARF proteins from other organisms. The entire nucleotide sequence of the potato ARF cDNA was obtained and found to display partial homology to the Cucurbita cDNA that was used as a screening probe (see Discussion). The potato clone contained a 637 bp long insert including an open reading frame of 591 nucleotides encoding a protein of 197 amino acids. The calculated molecular mass is 22.6 kDa. A rather short untranslated sequence was present at the 3" end of the cDNA and no poly(A)-tail was present (which, however, was frequently observed in the cDNA library used for screening and therefore does not appear to be a special feature of the ARF cDNA; Muller-Rober, unpublished observations). The Solanum tuberosum ARF cDNA sequence has been deposited in the EMBL/GenBank database under accession number X74461. A comparison between the potato ARF amino acid sequence with ARF proteins from other species is shown in Fig. 1. The sequences are highly conserved. When the potato ARF sequence was compared on the nucleotide level with other known sequences in the GenEMBL database, highest homology (78.4% identity in a 587 bp overlap) was found to the Arabidopsis thaliana ARF sequence. Very high homology was also found to several ARF sequences from cow (up to 79% identity in a 542 bp overlap), fungi (e.g. Candida albicans ARF1; 72.6% identity in 572 bp overlap) and man (76.1% identity in 549 bp overlap). Comparison of the deduced potato ARF amino acid sequence with sequences in the SwissProt databank revealed highest homology to the ARF1 protein from man (92.3% identity in 181 amino acid overlap). A lower degree
cDNA insert of plasmid a215 encoding a 14-3-3 protein (Ichimura et al. 1988) of Cucurbita (EMBL/GenBank accession number X76086) was used as a hybridization probe. Radioactive labelling was performed using a multiprime labelling kit (Boehringer Mannheim). Hybridization was done overnight at 42~ in 250 ram sodium phosphate buffer (pH 7.2) containing 7% (w/v) SDS, 1% (w/v) BSA and 1 mM EDTA. Filters were washed in 0.1 x SSC, 0.5% (w/v) SDS at 42 ~ C for 30 rnin. Plaque-purified phages were subjected to in vivo excision according to the manufacturer's (Stratagene) conditions. Rescued phagemid clones were investigated by restriction analysis and DNA sequencing. DNA sequencing and data analysis o.f potato ARF cDNA. DNA sequences were determined by the dideoxy method (Sanger et al. 1977). Commercial sequencing primers (Pharmacia, Freiburg, Germany) and specifically synthesized oligonucleotides (TibMolbiol, Berlin, Germany) were used for plasmid sequencing with T7 DNA polymerase (pharmacia). Sequence analysis was performed using the programs of the University of Wisconsin Genetics Computer Group (GCG Package, Version 7) (Devereux et al. 1984). Sequence comparisons on cDNA and deduced amino acid sequences were performed in GenBank, EMBL and SwissProt databases using the FASTA search program (Pearson and Lipman 1988). Northern blot analysis. Total RNA was prepared from frozen plant material using the guanidinium hydrochloride method as described by Logemann et al. (1987). Following electrophoresis (1.5% [w/v] agarose, 15% [v/v] formaldehyde) RNA w ~ transferred to nylon membranes (Hybond N, Amersham, UK). Membranes were hybridized overnight at 65~ in the buffer described in section describing cDNA library screening. The EcoRI cDNA insert of the plasmid PA215 (see Results) was used as a radioactively labelled hybridization probe. Filters were washed three times in 0.1 x SSC, 0.5% (w/v) SDS for 30 rain at 65~
Results Isolation and sequence analysis of a potato ARF cDNA
A potato leaf cDNA library was screened with a Cucurbita 14-3-3 protein cDNA probe under moderately stringent conditions as described in Materials and me~ods. Several A t a r f . Pep Boarf. Pep Hsa~f.Pep Scarf. Pep SCarf.Pep At:arf .Pep Boaxf.Pep H~axf.Pep Scaxf.Pep S~.arf.Pep A t a r f . Pep Soa~f.Pep llsar f .Pep Scarf.Pep Sl:arf .Pep
Atarf.Pep
Boarf.Pep Hsarf.Pep Scarf.Pep
Staff.Pep
I~A~Lr~alLF0~KZ
RIL
TISIKLPS]EL~L_.~AIKKEHR~
VG
DAA~KTT
VG
D.AAGKTTILTKLKL
LT~LK~
GGEEZVTTI ~ T EVITTI ~ GE V T T r P TTI
C
SO 50 50
GQDKIRPLWRHYTQHTQGLIPVVDSNDRDRV~ F~VETVETKNI FTVWDVGGQDKIRPLWRHYPQNTQGLIFV~DSNDASRV[ FNV~TV,TKNIS,TVWDVGGQDKIRPL,~HTFONT~GLIFV DS~DXERV[ , . v , v [ ] T x . Sr,vwDvc~QD@,~[]Lw S , , [ ] N , m C ~ , r V ~ D S D~RII FNVETVETKNI.SFTVWDVG ~DKIRPLWRHTFQNT~LIFV DSHDRORV|
I00
HEARS
ISO
I~HVETVSYKMI~FTVMDV g LSRMLAEDELXDAVLLVFANKQD
pgAM.AAEI
~sx~E~n~xxL~sss~x~xf'X-~vrx~x~P~x~xx~s , r. s ~ T x s ~ LSNQL~QK] NJWTIQAT T~GD EGLD NIWTIQATCAT G D G L T E G L D WL S N Q L R 14QKI PIWPIQATCATSGEGLTSGLE ,. s ~ t ~ HIWYTQ~TCATSGEGLTEGLD
LG
TD
~
HSLXIH
us~
I00
100
loo 100
~s0 lal
tSt
181 9
181
Fig. 1. Alignment of the deduced ARF amino acid sequence from Solanum tuberosum L and other species. The sequences are described in: Atarf - Arabidapsis thaliana (Regad et al. 1993); Boarf - cow (Sewell and Kahn 1988); Hsarf - man (Kahn et al. 1991); Scarf - Saccharomyces cerevisiae (Sewell and Kahn 1988); Staff -Solanum tuberosum (this report). Numbers indicate amino acid positions.
182 of homology was found to bovine ARF and several other human ARFs, as well as to ARFs from Saccharomyces cerevisiae (79.4% identity to ARF2 and 78.8% identity to ARF1), Giardia lamblia (68.6%) and chicken (67.4%). Appreciable homology to ARF-like proteins (Tamkun et al. 1991) of Drosophila melanogaster (56.6% identity) was also identified. Several conserved motifs, which are present in ARF proteins, are also observed in the potato ARF sequence shown here. Three consensus sequences proposed to be involved in GTP binding (Kaziro et al. 1991) are present at amino acid positions 24 - 31 (sequence GLDAAGKT; P motif of ARF proteins), 67 - 71 (sequence DVGGQ, G" motif DXXGQ), and 126 - 129 (sequence NKQD, G motif NKXD). ARF proteins usually have a length of 180 or 181 amino acids. Interestingly, potato ARF contains a Cterminal extension (16 amino acids long) which is not present in most of the other ARFs. The role of this extension is not known.
Expression analysis of potato ARF In order to investigate the mRNA expression of ARF, northern blot experiments were performed. Total RNA was isolated from various tissues of a collection of 4 independent 10 - 12 weeks old potato plants and after electrophoretic separation hybridized against the potato ARF cDNA probe. In all tissues tested, a ca. 0.9 kb long transcript was detected under high stringency conditions (Fig. 2). The strongest expression was found in growing tubers of different developmental stages, although the expression level varied between individual tubers. A strong expression was also seen in roots, whereas in sink and source leaves of the potato plants usually a lower level of mRNA was detected.
Discussion By screening a potato leaf cDNA library, we have identified a cDNA encoding a protein with high homology to ADP-ribosylation factor (ARF) proteins from a variety of other organisms, including animals and fungi. The ARF cDNA was identified in a heterologous screening procedure using a cucurbita eDNA encoding a 14-3-3 protein (Ichimura et al. 1988). When the deduced amino acid sequence of the potato ARF (see Fig. 1) was compared with the deduced amino acid sequence of the Cucurbita 14-3-3 protein no significant homology between the two proteins was found (not shown). However, a 49 bp long region of high homology (84%) was detected between the two cDNAs (not shown) which could explain the crosshybridization during the screening. Although ARF and 14-3-3 proteins do not have the same function, the recently published observation that the bovine FAS protein (a member of the 14-3-3 protein family) is able to activate
Fig. 2. Northernblot analysisof ARF gene expressionin varioustissuesof potato plants. Total RNA (501.tg/lane) was fractionated by denaturing agarosegel electrophoresisand aftertransferto nylonmembraneshybridized againstthe 32p-labelledpotatoARFeDNAisolatedfromclonePA215.T1, T2, T3: developingtubers of differentdevelopmentalstages; R: root; G: gynoeceum;St: stolon;So: sourceleaf;Si: sinkleaf. ADP-ribosylation (Fu et al. 1993) is interesting. The mRNA corresponding to the ARF cDNA described here is expressed in all tissues analysed. In addition, several experimental treatments, like incubation of detached leaves in light or darkness, or feeding of sucrose or nitrate to the leaves, did not significantly influence ARF mRNA levels (data not shown). Likewise, in Arabidopsis it has been shown that ARF mRNA expression during the growth cycle of a cell culture, including the stationary phase, was nearly constant. The function of ARF proteins in higher plants is not known at present. Since ARF expression is rather high in the various potato tissues and not strongly influenced by various environmental conditions (at least not in short term experiments, i.e. up to 48 h of treatment) it might be concluded that plant ARFs play a rather general (or housekeeping) function. No mutants deficient in ARF proteins have been described for higher plants. Loss of functional ARF proteins could be lethal to the plant, or the absence of ARF proteins is not accompanied by an easily visible phenotype. The cloning of the potato ARF eDNA described in this report will allow an alternative approach to investigate the role of ARF proteins: the cDNA can be used to down-regulate the levels of ARF mRNA (and thereby corresponding ARF protein) via the antisense RNA technique in transgenic plants (Van der Krol et al. 1988). Usually, antisense inhibition varies between independent transformants. Therefore, even in the case that complete deficiency in ARF proteins is lethal, it should be possible to recover transgenic plants which have only partially lost the
183 ARF protein. Preliminary data show that ARF mRNA levels can strongly be down-regulated in transgenic potato plants without severe changes in plant development (Szopa and Muller-Rober, unpublished data). The antisense plants hopefully will allow to assign a function to plant ARF proteins. Acknowledgement. This work was supported by Grant 400 88 91 01 from KI3N, and by the DFG.
References
Cassel D and Pfeuffer T (1978) Proc Natl Acad Sci USA 75:2669-2673 Devereux J, Haeberli P and Smithies O (1984) Nucl Acids Res 12:387-395 Duman RS, Winston SM, Clark JA and Nestler EJ (1990) J Neurochem 55:1813-1816 Fu H, Cobum J and Collier RJ (1993) Proc Natl Acad Sci USA 90:2320-2324 Ichimura T, Isobe T, Okuyama T, Takahashi N, Kazuaki A, Kuwano R and Takahashi Y (1988) Proc Natl Acad Sci USA 85:7084-7088 Kahn RA and Gilman AG (1986) J Biol Chem 261: 79067911 Kahn RA, Kern FG, Clark J, Gelmann EP and Rulka C (1991) J Biol Chem 266:2606-2614 Kaziro Y, Itoh H, Kozasa T, Nakafuko M and Satoh T (1991) Annu Rev Biochem 60:349-400 Kossmann J, Muller-Rober B, Dyer TA, Raines CA, Sonnewald U and Willmitzer L (1992) Planta 188: 712 Logemann J, Schell J and Willmitzer L (1987) Anal Biochem 163:21-26 Moss J and Vaughan M (1993) Cellular Signalling 5: 367379 Orci L, Palmer DJ, Amherdt M and Rothman JE (1993) Nature 364:732-734 Pearson WR and Lipman DJ (1988) Proc Natl Acad Sci USA 85:2444-2448 Regad F, Bardet C, Tremousaygue D, Moisan A, Lescure B and Axelos M (1993) FEBS Lett 316:133-136 Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press Sanger F, Nicklen S and Coulson AR (1977) Proc Natl Acad Sci USA 74:5463-5467 Schleifer LS, Kahn RA, Hanski E, Northup JK, Sternweis PC and Gilman AG (1982) J Biol Chem 257:20-23 Serafini T, Orci L, Amherdt M, Brunner M, Kahn RA and Rothman JE (1991) Cell 67:239-253 Sewell JL and Kahn RA (1988) Proc Natl Acad Sci USA 85:4620-4624 Stearns T, Kahn RA, Botstein D and Hoyt MA (1990a) Mol Cell Biol 10:6690-6699
Stearns T, Willingham MC, Botstein D and Kahn RA (1990b) Proc Natl Acad Sci USA 87:1238-1242 Tamkun JW, Kahn RA, Kissinger M, Brizuela B J, Rulka C, Scott MP and Kennison JA (1991) Proc Natl Acad Sci USA 88:3120-3124 Tsai S-C, Adamik R, Tsuchiya M, Chang PP, Moss J and Vaughan M (1991) J Biol Chem 266:8213-8219 Van der Krol A, Mol J and Stuitje A (1988) BioTechniques 6:958-976
