Polymerase Chain Reaction (Pcr) Primers for Amplifying Ribulose-1,5-Bisphosphate Carboxylase from Lichens

J. Ha/lori Bot. Lab. No. 88: 61 - 67 (Aug. 2000)

POLYMERASE CHAIN REACTION (PCR) PRIMERS FOR AMPLIFYING RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE FROM LICHENS

1 2 AURELIO REYES , MARIA DEL CARMEN MOLINA 3 AND CARLOS VICENTE '*

SUMMARY . Amplification products of both large and small subunits of rubisco have been construct ed with lichen thalli of C/adonia verticillaris and Evernia prunastri and their respective photobionts, using as primers oligonucleotides of Spinacea o/eracea (large) and Arabidopsis thaliana (small) genes. Although a pattern of multiple bands appeared by the southern analysis of the large as well as the small subunits, hybridation experiments revealed that only one band exclusively hybridized, in both cases, with the corresponding amplified oligonucleotide. KEY WORDS: C/adonia vertici//aris, Evernia prunastri, PCR, rubisco. ABBR EVIATIONS: dNTP: deoxy-nucleotide triphosphate; EDTA: ethylenediamine tetraacetic acid; lgG: immunoglobulin G; PBS: saline phosphate buffer; PCR: polymerase chain reaction; PVP: polyvinyl pyrrolidone; SOS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SSC: sodi um -sodium citrate buffer; TAE: Tris-(hydroxymethyl)-aminomethane-sodium-acetate-ethylene di amine tetracetic acid; Tris-HCl: Tris-(hydroxymethyl)-aminomethane hydrochloride.

I NTRODUCTION Chloroplast enzyme rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4. l . l .39) is a hexadecamer containing eight 53-kD large subunits and eight 14 kD small subunits. However, some primitive rubisco molecules from Rhodospirillum rubrum, Chlorobium thiosulfatophillum or Thiobacillus intermedius are only dimer, hexamer or oc tamer, respectively, of the large subunit (Akazawa, 1979). Small subunits are synthesized in the cytoplasm (Chua & Schmidt, 1978) whereas large subunits are synthesized in the chloroplast stroma (Blair & Ellis, 1974). Rubisco assembles within the chloroplast in a re action which implies the binding of the large subunit to a chloroplast chaperonin 60 (Goloubinoff et al., I 989) to be transferred on the small subunit in a reaction that requires ATP (Hubbs & Roy, 1993) The occurrence of rubisco in lichens has only been often supossed. Hill (I 976) de scribes lichens as, presumably, C3 plants on the basis of the ability of the lichen phyco bionts to produce monosaccharides and polyols. However, they must be considered as atyp-

1 Centro di Studio sui Mitocondri e Metabolismo Energetico. CNR. Via Amendola 165/A, 70 126 Bari, lta ly. 2 Department of Plant Biology II , Faculty of Pharmacy, Complutense University, 28040 Madrid, Spain. 3 Department of Plant Physiology, Faculty of Biology, Complutense University, 28040 Madrid, Spain. * To whom correspondence should be addressed. Fax number: 34-91-3945034; e-mail: [email protected] 62 J. Hattori Bot. Lab. No. 88 2 0 0 0

ical C3 plants. Ramalina maciformis, for example, has a C02 compensation point of about 1 6 µ11 - • This very low value indicates that glycine decarboxylation is not an immediate event after the oxigenating breakdown of ribulose-1,5-bisP but glycolate could be exported to the mycobiont to be metabolized at a rate independent on its production (Cowan et al., 1992). Rubisco has been described by Janson et al (1993) in the cyanobiont of Lichina con- .finis and by Ascaso et al. (1995) as a protein located in the pyrenoid matrix of Trebouxia from Umbilicaria cinereorufescens and Parmelia sulcata, although the apparent concentra tion of rubisco does not correlate with artificially induced changes in pyrenoid volume (Balaguer et al., 1996). This location is consistent with that of rubisco activase in the pyrenoid of green algae described by McKay et al. (1991 ). In this paper, conclusive evidence about the occurrence of rubisco in two lichen species is reported on the basis of PCR amplification of the oligonucleotide sequences of both large and small subunits and the corresponding hybridation probes.

MATERIAL AND METHODS Preparation of cell-free extracts Samples of 200 mg of thalli of C. verticillaris (Hoffm.) Schaer., from sandy soils of the tabuleiro of Joao Pessoa (NE Brazil) and Evernia prunastri (L.) Ach. on Quercus pyre naica branches from Valsain (Segovia, Spain) were rehydrated for 30 min. by floating them on 15 ml distilled water and afterwards they were macerated in a mortar with 10 ml 50 mM citrate-NaOH buffer, pH 8.0. Homogenates were centrifuged at l,350Xg for 5min. at 2°C. Supernatants were discarded and pellets resuspended in 2.0 ml citrate buffer and disrupted for 3 min at 2 kHz using a MSE sonic oscillator. The second homogenates were centrifuged at 16,600Xg for 15 min. at 2°C and suupernatants were used as cell-free extracts to sepa rate rubisco and to identify it by western blotting. Isolation ofalgal cells Phycobionts from E. prunastri and C. verticillaris were isolated according to the method of Ascaso ( 1980). After mechanical disruption of thalli in distilled water, suspen sions were centrifuged in a sucrose-Kl gradient. Algal cells, which remained in the inter phase, were collected and repeatedly washed with distilled water. Electrophoresis and immunoblotting SDS-PAGE and immunoblotting were performed with PhastSystem™ Pharmacia and a semidry electrophoretic transfer unit PhastTransfer™ according to the manufacturer's in structions. Samples were run in l 0- 15% polyacrylamide-gradient gels with 0.1 % SDS buffer. Gels had a 13 mm stacking zone and a 32 mm gradient zone. Proteins from gels after SDS-PAGE were electrophoretically transferred to nitrocellulose membranes. Trans fer buffer contained 25 mM Tris-HCI buffer, pH 8.0, 192 mM glycine and 20% methanol (v/v). Membranes were incubated with mouse rubisco antiserum (I: 1000 w/w) overnight at room temperature followed by an incubation for 3 h with rabbit antibody directed to mouse immunoglobulin G, and 3 h with goat-anti rabbit IgG covalently coupled to horse radish peroxidase (1 : lOOOw/w) in order to provide the corresponding visible reaction product. Bands on membranes were revealed by staining with protogold (BioCell Research A. REYES et al.: Rubisco from lichens 63

Lab.) after washing with 0.3% (v/v) Tween-PBS buffer, pH 7.0, for 30 min. DNA extraction Samples of I 00 mg of E. prunastri and C. vertici/laris thalli, as well as their dried phycobionts were ground in 1.5 ml eppendorf tubes with liquid nitrogen and 600 µI of the extraction buffer (0.2 M sucrose, 50 mM EDTA, I% SDS, I 00 mM Tris-HCl, pH 7.5, and 5% PVP) were added. After incubation for 20 min. at 60°C, 120 µI of 3.0 M potassium acetate, pH 5.2, were added and the tubes kept for 10 min. at -20°C. They were then centrifuged at 13,000 Xg for 10 min. at 2°C and the pellets discarded. Supematants were incubated with

RNase A (IOµg!µl) and T 1 (lOunits/µI) for 30min at 37°C and then one volume of buffered- saturated phenol : chloroform : isoamyl alcohol (25 : 24: 1 v/v) was added, cen trifuging the mixtures at 7,000Xg for lOmin. The organic phase was removed. The aque ous phase was newly extracted with one volume of chloroform:isoamyl alcohol (24: 1 v/v), centrifuged at 7,000Xg for I 0 min. and the supemantant mixed with one volume of iso propanol and kept at - 20°C for 30 min. After this, the mixture was centrifuged at 13 ,000 Xg for !Omin.; the pellet was dried in vacuum and the dry residue was resuspended in 50µ1 IOmM Tris-HCI containing l.OmM EDTA . Spinacia oleracea and Arabidopsis thaliana genomic DNA was extracted according to Delaporta et al. ( 1983). DNA quantification was achieved using a Beckman DU-70 spec trophotometer. PCR amplifications Based upon the sequence of the large (L) rubisco subunit from S. oleracea and the small (S) subunit from A. tha/iana obtained from EMBL database (accesion numbers V00168 and XI4564, respectively), specific primers were designed. Thus, the sequence of the oligonucleotide primers used from both Land S subunits were: L up : 5' -GCCCGTTGCTGGAGAAGAGAAAA L lw: 5'-CGTGAATACCGCCTGAAGCAA Sup: 5'-ATCACAAGCATTGGGGGAAGA S lw: 5' -ACTGGACTTGACGGGTGTTGT The amplification procedure took place in 25 µI 10 mM Tris-HCI buffer, pH 9.0, 50 mM KCI, 0.1 % Triton X-100, 2.0 mM MgCl2, 200 M of each dNTP, 20 µM of each primer, lOOmg template DNA and 1.25 units of Taq polymerase (Promega). Each mixture was overlaid with 50 µI white mineral oil to prevent evaporation. Samples for amplification were subjected to one cycle of 5 min. at 94°C; 40 repeats of the following thermal cycles were performed: I min. at 94°C, 2 min. at 55°C and 2 min. at 72°C, and finally of one cycle of 6. min at 72°C, on a PTC-100 MRJ Research thermocycler. PCR amplification products were separated on 1.5% agarose gels run in I XTAE (40mM Tris-acetate buffer, pH 7.8 containing 1.0 M EDTA), stained with ethidium bromide and observed by transillumination with ultraviolet light. Phage Xl 74 digested with Hal III was used as a molecular marker. Southern hybridation Amplification products obtained with DNA from E. prunastri and C. verticillaris and their phycobionts were transferred to Hybon N membranes (Amersham) by capillary south- 64 J. Hattori Bot. Lab. No. 88 2 0 0 0 em blotting. S. oleracea amplification product of the L subunit of rubisco, contammg 866 bp, and the 625 bp amplification product of the small subunit cluster of genes from A. thaliana were used as probes onto the southern transfers of the whole amplification prod ucts obtained with DNA of E. prunastri and C. verticillaris and their phycobionts. Probes obtained were recovered from agarose gels and purified using Genectean II (Bio 10 I) and labelled by random priming with DIG-11-dUTP using the Boehringer-Mannhein kit. Hy bridations were performed in 10 ml 5 X SSC containing 0.5 per cent SOS, at 65°C overnight. Filters were rinsed for 10 min. at 3 7°C in 100 ml of 0.30 M NaCl and 30 mM sodium citrate, pH 7 .0, containing 0.1 per cent SDS and then for 30 min. at 65°C in 100 ml of 15 mM NaCl and 1.5 mM sodium citrate, pH 7 .0, containing 0.1 per cent SOS. Colour re actions to detect probe DNA-target DNA hybrids were carried out following the instruc tions of the manufacturer.

R ESULTS AND DISCUSSION The results obtained from western blotting using two lichen species revealed multiple bands, only one of which corresponded to the L subunit ofrubisco (data not shown) and in dicated that polyclonal antibody generated non-specific bands. Thus, identification of ru bisco with polyclonal antibodies could produce artifacts even when they are used for im munocytochemical detection (Ascaso et al., 1995, 1998). The size of protein was estimated M 12 34 5 12345

Fig. I. Amplification products by PCR (left) of: lane I: Arabidopsis thaliana; lane 2: Cladonia verticillaris thallus; lane 3: Cladonia verticillaris photobiont; lane 4: Evernia prunastri thallus; lane 5: Evernia prunastri photobiont, using oligonucleotides from the gen of the small subunit of rubisco from A. thaliana. Southern hybridation (right) of the totallity of the amplification products from the different biological material, against the primer of A. thaliana. M = molecular markers from phage X 174 digested with Hal Ill. A. REY ES et al.: Rubisco from lichens 65 as 41 - 52 kDa, values that were in agreement with those obtained for higher plants (Akaza wa, 1979). The absence of the S subunit from lichen extracts could be explained in differ ent ways. It is possible that its epytope could be unrecognized by the polyclonal antibody or that the amount of this subunit was very much lower than that of the large one. Hovewer, the S subunit was detected by PCR and southern analysis. PCR of A. thaliana revealed a band of 625 bp corresponding to a cluster of four genes (Fig. 1, lane 1) . For lichens and their phycobionts, a pattern of multiple bands appeared by the southern analysis although hybridation experiments revealed that only one band exclusively hybridized with the ampli fied oligonucleotide, the size of which was 180 bp for C. verticillaris (Fig. I, lane 2) and 160 bp for E. prunastri (Fig. 1, lane 3). These results were almost identical to those ob tained for the corresponding phycobionts (Fig. 1, lanes, 4 and 5). The band of S subunit corresponded to a product which largely differed in size to that obtained from A. thaliana, possibly as a consequence of the occurrence of multiple introns in the genome of Ara bidopsis. (Krebbers et al., 1988). These results are in agreement with those reported by Ap pleby et al. (1998), who were able to obtain strong evidence from PCR analysis that the test M1 2 3 4 5 1 2 3 4 5

Fig. 2. Amplification products by PCR (left) of: lane I: Spinacea o/eracea; lane 2: C/adonia vertici/laris thallus; lane 3: C. vertici/laris photobiont; lane 4: Evernia prunastri thall us; lane 5: E. prunastri photobiont, using oligonucleotides from the gen of the large sub unit of rubisco from S. o/eracea. Southern hybridation (right) of the totallity of the amplifica tion products from the different biological material, against the primer of S. o/eracea. M = molecular markers from phage X 174 digested with Hal llI. 66 J. Hattori Bot. Lab. No. 88 2 0 0 0 loci for the small subunit of rubisco are polymorphic at the interspecies level. Southern analysis of PCR products showed only one band with sequential homology to the L subunit and a molecular size identical to that obtained for S. oleracea (866 bp), used as a control (Fig. 2). Hybridation of the amplification products demonstrated that C. verticillaris and E. prunastri phycobionts have the structural genes for both small and large subunits of rubisco.

A CKNOWLEDGEM ENTS This work was supported by a grant from the Direcci6n General de lnvestigaci6n Cientifica y Tecno16gica (Ministerio de Educaci6n y Ciencia, Spain), PB93 0092. We would like to thank Dr J. M. Franco, D. F. J. Gallego and Dr R. Linacero for their valuable help. We would like to thank Prof. M. R. D. Seaward for critical reading of the manuscript.

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