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Silene plastocyanin is fully functional in transgenic tobacco
Plant Science, 83 (1992) 45-54 Elsevier Scientific Publishers Ireland Ltd.
45
Silene plastocyanin is fully functional in transgenic tobacco Douwe de Boer a, Annemiek Wilmink a, Alice Lever a, Fons Cremers a, Paul Hooykaas b and Peter Weisbeek a UDepartment of Molecular Cell Biology and Institute of Molecular Biology, University of Utrecht, P.O. Box 80.056. 3508 TB Utrecht and bPlant Molec. Biol. Res. Group MOLBAS, University of Leiden, Clusius Laboratory. Wassenaarseweg 64, 2333 AL Leiden, ( Tile Netherlands)
(Received November 17th, 1991; accepted December 23rd, 1991) Transgenic tobacco plants, expressing the Silene pratensis (white campion) gene for the precursor of plastocyanin, were analysed with respect to the functionality of the gene product. The gene was found to be expressed in all tissues that were examined due to the strong and constitutive cauliflower mosaic virus 35S promoter. In green tissue the Silene protein was transported into chloroplasts and routed to the chloroplast lumen, where it was found processed to its mature size. In non-photosynthetic tissue the protein is transported into chromoplasts or leucoplasts. In plants grown in tissue culture the amount of endogenous tobacco plastocyanin was found to be reduced significantly, but the Silene plastocyanin was clearly detectable. When plants were dependent on photosynthesis for growth, due to depletion of sucrose from the medium, still only Silene plastocyanin was present. This strongly suggests that the Silene protein can take over photosynthesis in transgenic tobacco when the endogenous plastocyanin is not present. Silene plastocyanin is considered to be fully functional, since both its transport to the chloroplast and its function in photosynthesis were retained in transgenic tobacco plants. Key words." protein transport; plastid; transgenic plants; photosynthesis; norflurazon
Introduction The D N A present in m i t o c h o n d r i a a n d p l a s t i d s has a limited c o d i n g capacity. T h e r e f o r e these organelles rely for their p r o p e r f u n c t i o n i n g u p o n n u c l e a r - e n c o d e d p r o t e i n s synthesized in the cytoplasm. These p r o t e i n s are u s u a l l y m a d e as p r e c u r sor p r o t e i n s a n d are p o s t - t r a n s l a t i o n a l l y transp o r t e d into the organelles. P r o t e i n i m p o r t into m i t o c h o n d r i a has m a i n l y been s t u d i e d in a n i m a l cells, N e u r o s p o r a a n d yeast (reviewed by H a r t l et al. [1]) but recently also in p l a n t cells [2-5]. P r o t e i n i m p o r t into p l a s t i d s has m a i n l y been studied in vitro with i s o l a t e d p e a c h l o r o p l a s t s (reviewed by D e Boer a n d W e i s b e e k [6]). This imp o r t process is A T P - d e p e n d e n t [7] as is b i n d i n g o f the p r e c u r s o r to the c h l o r o p l a s t e n v e l o p e [8]. A Correspondence to: Douwe de Boer, Agrotechnological Research Institute (ATO-DLO), P.O. Box 17, 6700 AA Wageningen, The Netherlands.
specific s t r o m a l processing p e p t i d a s e (SPP) cleaves off the N - t e r m i n a l transit p e p t i d e when the precursor enters the s t r o m a [9]. S o m e n u c l e a r e n c o d e d p r e c u r s o r proteins, like p l a s t o c y a n i n a n d c o m p o n e n t s o f the oxygen evolving complex, a d d i t i o n a l l y have to cross the thyla k o i d m e m b r a n e system in o r d e r to reach their final location. I m p o r t o f these p r o t e i n s was shown to consist o f two distinct steps a n d the targeting signal o f the p r e c u r s o r has two s e p a r a t e d o m a i n s [10-12]. T h e F i r s t d o m a i n is c o m p a r a b l e to a transit p e p t i d e a n d is cleaved off by the SPP, resulting in an i n t e r m e d i a t e - s i z e d p r o t e i n present in the s t r o m a l fraction. The s e c o n d d o m a i n , the t h y l a k o i d transfer d o m a i n , is essential for thylakold lumen targeting [13] a n d is r e m o v e d by a p r o cessing p e p t i d a s e that is a s s o c i a t e d with the thyla k o i d s [14,15]. To study the t r a n s p o r t o f the t h y l a k o i d lumen p r o t e i n p l a s t o c y a n i n in vivo we t r a n s f o r m e d tobacco p l a n t s with the S i l e n e p r a t e n s i s p r e - p l a s t o -
0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
46
cyanin gene (pPC). The construct was placed under the control of the cauliflower mosaic virus 355 promoter to obtain constitutive expression in all tissues [16]. It was therefore also possible to examine protein transport in non-green tissues. The ability of Silene plastocyanin to take part in photosynthesis was studied in transgenic tobacco plants grown in tissue culture. These plants showed a significantly reduced level of the endogenous tobacco plastocyanin when the heterologous Silene plastocyanin was present. Materials and Methods
Plant transformation and tissue culture The gene for the Silene plastocyanin precursor (pPC) was cloned behind the cauliflower mosaic virus 35S promoter in the binary plant vector pBIN19 [17] as described previously [18]. The construct was conjugated into Agrobacterium tumefaciens LBA4404 containing the modified Tiplasmid pAL4404 [19] in a triparental mating event using the helper plasmid pRK2013 [20]. The construct was introduced into Nicotiana tabacum var. Petit Havanna (SR1) using the leaf disc transformation method [39]. Regenerated plants were selected for kanamycin resistance and grown on tissue culture medium or on soil. The individual transformed plants were numbered A2-1 to A2-8. Plants were grown in tissue culture in small plastic containers with Murashige and Skoog medium without plant hormones [21]. When sucrose was not included in the tissue culture medium, small holes were made in the lid of the containers to optimize diffusion of gases. The lid was subsequently covered with sterile filter paper to prevent infection. To minimize the amount of nutritive matter present in the transferred plants, we only transferred small pieces of stem with one internode to the sucrose-depleted medium. When required, noflurazon was present in the medium at a concentration of 20 ~M. Nucleic acid isolation and blotting D N A was isolated from plants grown on soil using a modified CTAB procedure [22]. Young leaves (about 10 g) were ground in liquid nitrogen and suspended in 10 ml of 2% CTAB, 1.4 M NaC1,
100 m M Tris-HC1 (pH 8.0), 20 m M E D T A and 2% 13-mercaptoethanol. After 30 min incubation at 55°C the suspension was chloroform extracted twice. One tenth volume of 10% CTAB and 0.7 M NaC1 was added before the second extraction. An equal volume of 1% CTAB, 50 m M Tris-HC1 (pH 8.0) and 10 mM E D T A was added and the suspension was incubated for 30 min at room temperature. After centrifugation at 4000 rev./min ( ± 2000 x g) in an SS35 rotor the pellet was washed once with 70% ethanol, resuspended in a CsCI solution and further purified by density centrifugation as described by Maniatis et al. [23]. D N A concentrations were obtained spectrophotometrically assuming that 1 OD260 = 50 t~g DNA/ml. Restriction enzyme digestions, Southern transfer and hybridizations were according to Maniatis et al. [231. R N A was isolated from young leaves of plants grown in soil or in tissue culture by the modified method of Chirwin et al. [24]. Two grams of leaf tissue were ground in liquid nitrogen and 8 ml of 4 M guanidine thiocyanate, 5 m M Na3-Citrate, 0.5% sarkosyl, 0.3% antifoam and 0.1 M 13mercaptoethanol was added. After centrifugation for 10 min in an SS34 rotor at 10 000 rev./min ( + 1 2 000 × g) 1 g/ml CsCi was added to the supernatant. This suspension was transferred to a centrifuge tube on top of a layer of 3 ml 5.7 M CsCI and 0.1 M E D T A (pH 7.5). After centrifugation for 23 h at 29 000 rev./min ( ± 140 000 × g) in an SW41 rotor the pellet was resuspended in 400/A of 10 mM T r i s - H C l (pH 7.5), 5 mM E D T A and 0.!% SDS and extracted twice with butanol/ chloroform (1:4). After ethanol precipitation R N A concentrations were obtained spectrophotometrically assuming that 1 0026o = 40 tzg R N A / ml. R N A electrophoreses and Northern blotting were done using the formaldehyde gel system described by Maniatis et al. [23].
Protein isolation and Western blots Total protein was isolated by grinding tissue in liquid nitrogen and extracting the powder with 30 mM potassium phosphate (pH 7.5), 400 mM NaC1, 2 mM 13-mercaptoethanol, 100 I~M P M S F and 1% Triton X-100. After determining the amount of protein [25], samples of 20 izg protein
47
were electrophoretically separated on a 15% SDS polyacrylamide gel system and blotted as described previously [18].
pBIN 19 RB - - 1
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.
Chloroplast isolation and fractionation Chloroplasts were isolated from tobacco leaf tissue as described for pea leaves [26]. To remove external proteins the chloroplasts were treated with 0.1 mg/ml thermolysin for 30 min at 4°C. The reaction was stopped by adding EDTA to 10 mM and reisolation of the intact chloroplasts by centrifugation through a 40% Percoll cushion [26]. The chloroplasts were fractionated after they were lysed in 50 mM Hepes (pH 8.0) by spinning down the thylakoids (10 min at 4000 x g). The thylakoids were washed once with 50 mM Hepes (pH 8.0) and 330 mM sorbitol and incubated with 0.4 mg/ml thermolysin for 30 min at 4°C to remove contaminating proteins. The thermolysin treatment was stopped by adding EDTA to 10 mM and reisolation of the thylakoids. Thylakoids were separated in a lumen and a membrane fraction. To achieve this the thylakoids were sonicated after repeated freezing and thawing and the membranes were pelleted by centrifugation for 30 min at 40 000 x g.
Immuno-gold labelling Ultrathin cryosections [27] of tissue of tobacco plants were incubated with antibodies against spinach plastocyanin [28] and labelled with 9 nm protein-A gold particles. Results
Plant transformation and analysis of expression The Silene plastocyanin gene [28] was cloned behind the 35S cauliflower mosaic virus promoter in the binary plant transformation vector pBIN19 [17]; this construct, pBIPC7, is shown in Fig. 1. Transformation of tobacco plants with this construct resulted in eight regenerated kanamycin resistant plants and these were analysed. The presence of the Silene plastocyanin gene in the tobacco genome was determined with a Southern analysis of EcoRI digested chromosomal DNA (Fig. 2a). When the coding sequence of the Silene plastocyanin transit peptide was used as a probe,
plastocyanin transit ~- .............
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Fig. 1. The plastocyanin expression plasmid (pBIPC7) used to transform tobacco plants. The construct contains the plastocyanin coding sequence behind the 35S promoter cloned in the EcoRI site of the MCS of the vector pBIN 19 in the orientation indicated. LB and RB denote left and right border of T-DNA, respectively, pr.NOS, nopaline synthetase promoter; t.NOS, nopaline synthetase terminator: NPT, neomycin phosphotransferase gene; MCS, multiple cloning site: c~-LACZ, alpha complementary region of the ~-galactosidase gene: E, EcoR1; H, HindIll.
the expected 1.8 kb EcoRI fragment, containing the 35S-promoter and the plastocyanin coding sequence (see Fig. 1), was detected in the genomic DNA of all transgenic plants. Besides a very faint 1.8-kb band a much larger hybridizing fragment is present in the digested DNA of plant A2-3. The band with the higher molecular weight can be explained when loss of one of the two EcoRI sites in this copy of the integrated DNA is supposed. No cross hybridization with the endogenous tobacco genes or with vector DNA (pBINI9) was seen under the applied stringency (Fig. 2a). The number of integrations into the tobacco genome was estimated by densitometer scanning of the 1.8-kb band using specified amounts of the EcoRI digested plasmid pBIPC7 as a reference. During the calculations a haploid genome size of 1.52 x 109 bp [29] was assumed for tobacco. The number of intact insertions per diploid genome varied from one in A2-3 to eight in A2-4 (see Table I). Transcription of the introduced gene was probed by Northern analysis of leaf RNA. Transcripts were detected in all plants except A2-3 (Fig. 2b).
48
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Fig. 2. Analysis of transgenic plants grown in soil. (A) Southern blot of EcoRl-digested chromosomal DNA. Tobacco (SR1) and Silene chromosomal DNA and vector pBINI9 DNA were used as a control. Ten micrograms of digested DNA is applied per lane. One genome equivalent (a) and five genome equivalents per haploid genome (b) of plasmid pBIPC7 were loaded as references. A Hindlll/Ncol fragment from pPCS74 [10] coding for the transit peptide of plastocyanin was used as a probe. Molecular weight markers are in kbp. (B) Northern blot of total RNA isolated from leaf tissue. RNA isolated from Silene and tobacco (SR I ) leaf tissue was used as a control. Forty micrograms of RNA was applied per lane. Probe and size markers are as described in (A). (C) Western blot of protein isolated from leaf tissue. Protein isolated from leaf tissue of Silene and non-transformed tobacco was used as a control. Twenty micrograms of protein was applied per lane. Antibodies raised against spinach plastocyanin [28] were used to detect plastocyanin. Molecular weight markers are in kDa.
The RNA level in the different plants is not exactly comparable with the number of inserts per individual plant (see Table I), e.g. A2-5 has a higher RNA level than A2-4 although the number of inserts in the genome is lower. Position effects may well be the cause of these differences. The size of the transcripts in all plants was comparable to the size of the plastocyanin transcript in Silene plants (about 780 bp). This would be expected when normal transcription termination and polyadenylation of the introduced gene occurs in the transgenic tobacco plants. Cross hybridization with endogenous plastocyanin transcripts was not observed under these conditions. Translation of the transcripts in tobacco leaf tissue of plants grown in soil was analysed in protein blotting experiments using antibodies directed against spinach plastocyanin (Fig. 2c). Control tobacco plants show two endogenous plastocyanin
bands, which both react with the plastocyanin antibody. Six out of eight plants tested show, besides the two endogenous plastocyanin bands, a new protein band that has the same mobility as the Table 1.
Genomic copy numbers and RNA expression levels of the Silene plastocyanin construct in the transgenic plants. Plant no.
Estimated copy no. per diploid genome
Relative amount of RNA (%)~
A2-1 A2-2 A2-3 A2-4 A2-5 A2-6 A2-7 A2-8
6 2 1 8 5 2 2 4
31.1 17.4 0.0 48.4 100.0 11.1 48.4 28.5
aThe RNA levels are given as percentages of the amount found in A2
49
mature Silene protein. Although we did not quantify the Western blot it is apparent that there is a correlation between the transcript level of the Silene gene and the amount of Silene plastocyanin detected. Plant A2-6 expresses the protein at a low level; overloading the gel is necessary to visualize the plastocyanin band. This plant also has a low level of transcription. The Silene protein could not be detected in plant A2-3 as could be expected from the Northern blotting experiments. There is apparently no effect of the presence of the Silene plastocyanin on the level of the endogenous tobacco plastocyanin in these plants, grown in soil. All eight plants were morphologically equal to wild type (SR 1) plants. In the non-green petal tissue of flowers, endogenous tobacco plastocyanin is not expressed. However, the Silene plastocyanin protein was shown to be present in petal tissue when assayed with protein blotting experiments (Fig. 3). All transformed plants with expression in leaf tissue also show expression in petal tissue. The same holds for root tissue, although the amount of Silene protein is low as compared to petal tissue (not shown). Cellular localization of Silene plastocyanin The Silene plastocyanin protein synthesized in leaf tissue of transgenic tobacco plants migrates with the same mobility and therefore has the apparent molecular weight of mature plastocyanin isolated from Silene leaves. The Silene precursor protein is apparently processed to its mature size. The localization of the Silene protein in the tobacco leaf cell was determined by isolation and fractionation of chloroplasts of the transgenic plant A2-5. The Western blot of the various fractions shows that both Silene and tobacco plastocyanin behave identically during fractionation (Fig. 4). Both proteins are protected against protease treatment of the intact chloroplast or the intact thylakoids and fractionate only in the thylakoid lumen fraction. Electron microscopical analysis of immuno-gold labelled coupes of leaf tissue also localizes all plastocyanin exclusively to the thylakoids (not shown). The Silene protein in petal and root tissue has the same electrophoretic mobility as mature plastocyanin in leaf tissue (see Fig. 3). The protein
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Fig. 3. Western blot of total protein isolated from petal tissue. Twenty micrograms of total protein isolated from petal tissue of tobacco plants transformed with pBIPC7 was used. Silene leaf and non-transformed tobacco (SR1) leaf and petal proteins were used as controls. Antibodies as indicated in Fig. 2c. 1, leaf protein; p, petal protein.
is therefore expected to be transported into the plastids present in these tissues. Electron microscopy in combination with immuno-gold labelling shows that in petal tissue plastocyanin is indeed present inside the chromoplasts (not shown). Only a limited amount of background staining is observed outside the plastids. Expression of plastocyanin in plants grown in tissue culture In transgenic plants analysed after growth in soil, no changes in the level of endogenous plastocyanin was observed (Fig. 2c). However, with plants that were permanently grown in tissue culture the amount of endogenous plastocyanin was found to be reduced significantly, whereas the amount of the Silene protein in most plants was unchanged. Figure 5a shows a Western blot of total protein isolated from such plants. A2-1 and A2-8, two plants that express the Silene plastocyanin gene at about the same level in tissue culture as when grown in soil (Fig. 2c), show a striking change in the level of endogenous tobacco plastocyanin. The amount of it was reduced by at least a factor ten. A faint signal of endogenous plastocyanin could be detected after overloading the gel. The quantity of other chloroplast proteins was not reduced, as was checked by Coomassie blue staining of total leaf protein after elec-
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Fig. 4. Localization of Silene plastocyanin in chloroplasts of the transgenic tobacco plant A2-5. (A) Western blot of total proteins isolated from chloroplasts. Protein isolated from Silene and non-transformed tobacco (SRI) leaves was used as a control. PC antibodies as indicated in Fig. 2c. 1, leaf protein; c, chloroplast protein. Arrows point at the position of mature Silene plastocyanin. (B) Western blot of total protein isolated from fractionated chloroplasts. 1, leaf protein; t, total thylakoid protein; tl, thylakoid lumen protein; tm, thylakoid membrane protein. Antibodies, controls and arrows as in (A).
trophoresis on SDS-PAGE (not shown). This absence of tobacco plastocyanin in tissue culture could be a consequence of the growth condition or of the presence of the Silene protein. To test this a non-transformed tobacco plant and the transgenic plants A3-3 and A3-6, which were transformed with a different gene cloned in the same vector, were also analysed. The reduction of the endogenous plastocyanin was not observed with these plants when grown in tissue culture (Fig. 5a). Therefore the effect, although only observed in
plants grown in tissue culture, is correlated with the presence of the Silene protein. This strong reduction of tobacco plastocyanin raises the interesting question whether the Silene plastocyanin can functionally replace the tobacco plastocyanin in photosynthesis. However, this question could not directly be answered, although these plants were able to grow in tissue culture medium in the absence of endogenous plastocyanin. Due to the presence of sucrose in the tissue culture medium these plants did not need to photosynthesize in order to grow. This was clearly shown when we included in the medium 20 ~zM norflurazon, an inhibitor of carotenoid biosynthesis, which leads to photo bleaching in the light [30]. Although the plants that were grown in this medium were white, they were able to grow heterotrophically (Fig. 6). Photosynthetically active plants growing in tissue culture were obtained by the exclusion of sucrose from the medium. Both plants A2-1 and A2-8 and the control plants were able to grow on sucrose-depleted medium. They both grew with a reduced growth rate and they were comparable to the green plants grown on a medium with sucrose (Fig. 6). Inclusion of 20 #M norflurazon in the medium without sucrose
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Fig. 5. Western blot of total protein isolated from plants grown in tissue culture. (A) Total protein isolated from leaf tissue of plants grown on tissue culture medium with sucrose. (B) As (A) but plants were grown on tissue culture medium without sucrose. For use as controls, protein was isolated from non-transformed tobacco (SR1) and two tobacco plants (A3-5 and A3-6), that were transformed with an arbitrary pBINI9 construct. These control plants were also grown in tissue culture• Protein isolated from Silene leaf tissue was used as a reference for mature Silene plastocyanin. Antibodies were as indicated in Fig. 2c. Molecular weight markers are in kDa. Arrows point at the position of mature plastocyanin.
51
Fig. 6. Tobacco plants grown in tissue culture media of different composition. The green (dark) plant at the left was grown on normal tissue culture medium with sucrose. The white plant on the right was grown on tissue culture medium with sucrose and 20 ~M norflurazon. The small white plants in the middle were grown on tissue culture medium without sucrose in the presence of 20/~M norflurazon.
resulted in very small white plants which died after a few weeks (Fig. 6). This convincingly showed that photosynthetic activity was necessary on a medium without sucrose. Reduced levels o f endogenous plastocyanin were found in tobacco plants expressing Silene plastocyanin, likewise whether they were grown in tissue culture medium with sucrose or without sucrose (Fig. 5b). A higher level o f endogenous plastocyanin would have been expected in case o f an inactive Silene plastocyanin protein. Therefore, we conclude that Silene plastocyanin is able to take part in photosynthesis in these transgenic tobacco plants. We can not exclude the possibility that the residual endogenous plastocyanin is taking part in photosynthesis, but we consider it unlikely that this small a m o u n t is sufficient for growth on a medium without sucrose.
Discussion We have obtained transgenic t o b a c c o plants
that properly express the Silene plastocyanin gene. The Silene plastocyanin protein could be separated from the two endogenous ones because o f differences in electrophoretic mobility on SDSP A G E . Our gelelectrophoresis system revealed the presence o f two endogenous plastocyanin bands in tobacco cells. Last and G r a y [31] only detected one band with a non-denaturing gelelectrophoresis system. The presence o f two different plastocyanin proteins is, however, very likely considering the allo-tetraploid nature o f N. tabacum [32]. Expression of the Silene plastocyanin protein did not affect the level o f endogenous plastocyanin when the plants were grown in soil. Similar results were also found in other studies in which foreign plastocyanin proteins were expressed in transgenic plants [18,31]. The Silene protein is imported into chloroplasts and routed towards the thylakoid lumen, where it is processed to its mature size. This means that the transit peptide o f the Silene protein is fully recognized by the tobacco protein transport ma-
52 chinery. Similar results were obtained with tomato plants transformed with the Silene plastocyanin gene [18]. Therefore it is likely that the mechanism of chloroplast protein import and routing is conserved among higher plants. Small differences will probably only affect the efficiency of the transport process. This view is also supported by in vitro import experiments with isolated pea chloroplasts. These pea chloroplasts are able to import proteins from a wide range of different plants (for an overview see Ref. 16). Due to the constitutively expressed CaMV 35S promoter Silene plastocyanin is also synthesized in petal and root tissue of the six different transgenic plants that were analysed. The endogenous plastocyanin is not made in non-green tissue. Silene plastocyanin is localized inside the plastids in these tissues, even though they are morphologically distinct from chloroplasts. Similar observations were made when Silene plastocyanin was expressed in various tissues of transgenic tomato plants [18]. Non-green plastids were also found to be competent for import of chloroplast proteins in in vitro import experiments [33-35]. Also amyloplast precursor proteins can be imported into chloroplasts in vitro [36]. In both tomato and tobacco the Silene plastocyanin protein was found processed to its mature size in these non-green tissues, although a real thylakoid membrane system is not present in the organelles. Experiments in which the precursor of plastocyanin was imported into castor bean leucoplasts in vitro showed the presence of an intermediate sized protein in the organelle [35]. This indicates that in vivo the presence of a mature-sized protein might reflect the situation after an additional aspecific processing. The mature part of the protein is probably more resistent to protease when folded properly after uptake of Cu >. Uptake of Cu 2+ can occur whenever mature plastocyanin is formed. Apoplastocyanin is shown to take up Cu 2+ in vitro without the help of a protein factor [37], and plastocyanin also incorporates Cu 2+ when it is mistargeted to the stroma in in vitro import experiments [38]. Therefore the mature part of the protein is expected to be selectively stabilized. This shows that it might be wrong to conclude that a protein is imported into organelles only by the observation that the precursor is processed.
We now can extend our previous conclusions [18] to the more general statement that the protein import machineries of different plastids in plants do not discriminate between proteins destined for either of them. Differentiation of the plastids during development seems to have no influence on the mechanism of protein import. If all different plastids are indeed capable of importing all the proteins present in either of them, it is difficult to believe that they possess a wide range of specific receptor proteins, one for each protein imported. For this reason it is more likely that there is only one receptor, or at most a few, with low specificity. In contrast to plants grown in soil, transgenic tobacco plants grown in tissue culture show a highly reduced level of endogenous plastocyanin when the Silene protein is present. This decrease in endogenous plastocyanin made the Silene protein the prevailing plastocyanin in these plants. This made it possible to study whether a foreign plastocyanin protein is able to take over electron transfer in the thylakoid lumen in vivo. During growth conditions that required photosynthesis we observed growth of both transgenic and control tobacco plants. The transgenic plants still only expressed Silene plastocyanin under these conditions, suggesting that this plastocyanin is active in photosynthesis in vivo. It is not completely excluded that the very low amount of endogenous plastocyanin, present in these plants accounts for all the photosynthetic activity. However, we consider this unlikely since no obvious differences were observed between control plants, which expressed tobacco plastocyanin at normal levels, and the transgenic plants, which only contained trace amounts of endogenous plastocyanin. We do not know why the endogenous plastocyanin protein is down-regulated in plants grown in tissue culture and not in plants grown in soil. It is possible that the presence of the Silene protein down regulates the endogenous plastocyanin level, but this down regulation is not sufficient when there is a strong demand for the protein, e.g. during rapid growth on soil. Measurements on pea plastocyanin isolated from transgenic tobacco plants showed that this foreign plastocyanin protein was correctly folded and able of electron transfer in vitro [31]. Taken together we can conclude that a plastocyanin gene
53 that duce tein that
is e x p r e s s e d in a f o r e i g n p l a n t is a b l e t o a functional protein capable of proper transport towards the thylakoid lumen t h i s p r o t e i n is a l s o a b l e t o p a r t i c i p a t e in
proproand elec-
11
tron transfer during photosynthesis. This proper f u n c t i o n i n g is e s s e n t i a l f o r in v i v o s t u d i e s w i t h a u t h e n t i c o r m u t a n t p r o t e i n s in t r a n s g e n i c p l a n t s .
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Acknowledgements We thank Dr. Sjef Smeekens for his participation and Mr. Harry Bettenbroek and Lucy Molen-
13
dijk for technical assistance during of this work. We are grateful to SANDOZ A.G. in Basel for his azon. This work was supported
14
Netherlands Organization Pure Science (SON/ZWO).
the initial Parts D r . J. H a r r o f gift o f n o r f l u r in P a r t b y t h e
for Advancement
of 15
References 1 F.-U. Hartl, N. Planner, D.W. Nicholson and W. Neupert, Mitochondrial protein import. Biochim. Biophys. Acta, 988 (1989) 1-45. 2 M. Boutry, F. Nagy, C. Poulsen, K. Aoyagi and N.H. Chua, Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants. Nature, 328 (1987) 340-342. 3 J. Whelan, L. Dolan and M.A. Harmey, Import of precursor proteins into Vicia faba mitochondria. FEBS Lett., 236 (1988) 217-220. 4 J.A. White and J.G. Scandalios, Deletion analysis of the maize mitochondrial superoxide dismutase transit peptide. Proc. Natl. Acad. Sci. U.S.A. , 86 (1989) 3534-3538. 5 U.K. Schmitz and D.M. Londsdale, A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo. Plant Cell, 1 (1989) 783-791. 6 A.D. de Boer and P.J. Weisbeek, Chloroplast Topogenesis: Protein import, sorting, and assembly. Biochim. Biophys. Acta, 1071 (1991) 221-253. 7 S.M. Theg, C. Bauerle, L.J. Olsen, B.R. Selman and K. Keegstra, Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes. J. Biol. Chem., 264 (1989) 6730-6736. 8 L.J. Olsen, S.M. Theg, B.R. Selman and K. Keegstra, ATP is required for the binding of precursor proteins to chloroplasts. J. Biol. Chem., 264 (1989) 6724-6729. 9 C. Robinson and R.J. Ellis, Transport of proteins into chloroplasts: partial purification of a chloroplast protease involved in the processing of imported precursor polypeptides. Eur. J. Biochem., 142 (1984) 337-342. 10 S. Smeekens, C. Bauerle, J. Hageman, K. Keegstra and P. Weisbeek, The role of the transit peptide in the routing of
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precursors toward different chloroplast compartments. Cell, 46 (1986) 365-376. J. Hageman, C. Baecke, M. Ebskamp, R. Pilon, S. Smeekens and P. Weisbeek, Protein import into and sorting inside the chloroplast are independent processes. Plant Cell, 2 (1990) 479-494. H.E. James, D. Bartling, J.E. Musgrove, P.M. Kirwin and R.G. Herrmann, Transport of proteins into chloroplasts import and maturation of precursors to the 33-kda 23-kda and 16-kda proteins of the photosynthetic oxygenevolving complex. J. Biol. Chem., 264 (19891 19573-19576. D. de Boer, H. Bakker, A. Lever, T. Bouma, E. Salentijn and P. Weisbeek, Protein targeting towards the thylakoid lumen of chloroplasts: proper localization is only observed in vivo. EMBO J., 10 (1991) 2765-2772. J. Hageman, C. Robinson, S. Smeekens and P. Weisbeek. A thylakoid processing protease is required for complete maturation of the lumen protein plastocyanin. Nature. 324 (1986) 567-569. P.M. Kirwin, P.D. Elderfield, R.S. Williams and C. Robinson, Transport of proteins into chloroplasts organization orientation and lateral distribution of the plastocyanin processing peptidase in the thylakoid network. J. Biol. Chem., 263 (1988) 18128-18132. P.R. Sanders, J.A. Winter, A.R. Barnason, S.G. Rogers and R.T. Frayley, Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants. Nucl. Acids Res., 15 (19871 1543-1558. M. Bevan, Binary Agrobacterium vectors for plant transformation. Nucl. Acids Res., 12 (1984) 8711-8721. D. de Boer, F. Cremers, R. Teertstra, L. Stairs, J. Hille, S. Smeekens and P. Weisbeek, ln-vivo import of plastocyanin and a fusion protein into developmentally different plastids of transgenic plants. EMBO J., 7 (1988} 2631-2636. A. Hoekema, P.R. Hirsch, P.J.J. Hooykaas and R.A. Schilperoort, A binary plant vector strategy based on separation of vir and T-region of the Agrobacterium tumejaciens Ti-plasmid. Nature, 303 (1983) 179-180. S.T. Lain, B.S. Lam and G. Strobel, A vehicle for the introduction of transposons into plant-assiciated pseudomonads. Plasmid, 13 (1985) 200-204. T. Murashige and F. Skoog, A revised medium for rapid growth and bio assay with tobacco tissue culture. Plant Physiol., 15 (1962) 473-497. M.G. Murray and W.F. Thompson, Rapid isolation of high molecular weight plant DNA. Nucl. Acids Res., 8 (1980) 4321-4324. T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1982. J.M. Chirwin, A.E. Przybyla, R.J. MacDonald and W.J. Rutter, Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18 (1979) 5294-5299. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing
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the principle of protein-dye binding. Anal. Biochem., 72 (1976) 248-254. K. Cline, M. Werner-Washburne, T.H. Lubben and K. Keegstra, Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts. J. Biol. Chem.~ 260 (1985) 3691-3696. J. Boonstra, P. van Maurik, L.H.K. Defize, S.W. de Laat, J.L.M. Leunissen and A.J. Verkley, Visualization of epidermal growth factor receptor in cryosections of cultured A431 cells by immunogold labeling. Eur. J. Cell Biol., 36 (1985) 209-216. S. Smeekens, M. de Groot, J. van Binsbergen and P. Weisbeek, Sequence of the precursor of the chloroplast thylakoid lumen protein plastocyanin. Nature, 317 (1985) 456-458. J.L. Zimmerman and R.B. Goldberg, DNA sequence organization in the genome of Nicotiana tabacum. Chromosoma, 59 (1977) 227-252. P.G. Bartels and C. McCullough, A new inhibitor of carotenoid synthesis in higher plants: 4-chloro-5-(dimethylamino)-2-~,~,~,(trifluoro-m-tolyl)-3(2H)pyridazinone (Sandoz 6706). Biochim. Biophys. Acta, 48 (1972) 16-22. D.I. Last and J.C. Gray, Synthesis and accumulation of pea plastocyanin in transgenic tobacco plants. Plant Mol. Biol., 14 (1990) 229-238. J.C. Gray, S.D. Kung and S.Q. Wildman, Origin of Nicotiana tabacum L. detected by polypeptide composition. Nature, 252 (1974) 226-227.
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S.A. Boyle, S.M. Hemmingsen and D.T. Dennis, Uptake and processing of the precursor to the small subunit of ribulose 1,5-bisphosphate carboxylase by leucoplasts from the endosperm of developing castor oil seeds. Plant Physiol., 81 (1986) 817-822. K. Strzalka, J. Ngernprasirtsiri, A. Watanabe and T. Akazawa, Sycamore amyloplasts can import and process precursors of nuclear encoded chloroplast proteins. Biochem. Biophys. Res. Commun., 149 (1987) 799-806. C. Halpin, J.E. Musgrove, J.M. Lord and C. Robinson, Import and processing of proteins by castor bean leukoplasts. FEBS Lett., 258 (1989) 32-34. R.B. Klo~gen, H. Saedler and J.-H. Weil, The amyloplasttargeting transit peptide of the waxy protein of maize also mediates protein transport in vitro into chloroplasts. Mol. Gen. Genet., 217 (1989) 155-161. T. Hibino, A.D. de Boer, P.J. Weisbeek and T. Takabe, Reconstitution of mature-plastocyanin from precursor apo-plastocyanin expressed in Escherichia coli. Biochim. Biophys. Acta, 1058 (1991) 107-112. H.M. Li, S.M. Theg, C.M. Bauerle and K. Keegstra, Metal-ion-center assembly of ferredoxin and plastocyanin in isolated chloroplasts. Proc Natl. Acad. Sci. U.S.A., 87 (1990) 6748-6752. R.B. Horsch, J.E. Fry, N.L. Hoffman, D. Eichholtz, S.G. Rogers and R.T. Frayley, A simple and general method for transferring genes into plants. Science, 227 (1985) 1229-1232.
45
Silene plastocyanin is fully functional in transgenic tobacco Douwe de Boer a, Annemiek Wilmink a, Alice Lever a, Fons Cremers a, Paul Hooykaas b and Peter Weisbeek a UDepartment of Molecular Cell Biology and Institute of Molecular Biology, University of Utrecht, P.O. Box 80.056. 3508 TB Utrecht and bPlant Molec. Biol. Res. Group MOLBAS, University of Leiden, Clusius Laboratory. Wassenaarseweg 64, 2333 AL Leiden, ( Tile Netherlands)
(Received November 17th, 1991; accepted December 23rd, 1991) Transgenic tobacco plants, expressing the Silene pratensis (white campion) gene for the precursor of plastocyanin, were analysed with respect to the functionality of the gene product. The gene was found to be expressed in all tissues that were examined due to the strong and constitutive cauliflower mosaic virus 35S promoter. In green tissue the Silene protein was transported into chloroplasts and routed to the chloroplast lumen, where it was found processed to its mature size. In non-photosynthetic tissue the protein is transported into chromoplasts or leucoplasts. In plants grown in tissue culture the amount of endogenous tobacco plastocyanin was found to be reduced significantly, but the Silene plastocyanin was clearly detectable. When plants were dependent on photosynthesis for growth, due to depletion of sucrose from the medium, still only Silene plastocyanin was present. This strongly suggests that the Silene protein can take over photosynthesis in transgenic tobacco when the endogenous plastocyanin is not present. Silene plastocyanin is considered to be fully functional, since both its transport to the chloroplast and its function in photosynthesis were retained in transgenic tobacco plants. Key words." protein transport; plastid; transgenic plants; photosynthesis; norflurazon
Introduction The D N A present in m i t o c h o n d r i a a n d p l a s t i d s has a limited c o d i n g capacity. T h e r e f o r e these organelles rely for their p r o p e r f u n c t i o n i n g u p o n n u c l e a r - e n c o d e d p r o t e i n s synthesized in the cytoplasm. These p r o t e i n s are u s u a l l y m a d e as p r e c u r sor p r o t e i n s a n d are p o s t - t r a n s l a t i o n a l l y transp o r t e d into the organelles. P r o t e i n i m p o r t into m i t o c h o n d r i a has m a i n l y been s t u d i e d in a n i m a l cells, N e u r o s p o r a a n d yeast (reviewed by H a r t l et al. [1]) but recently also in p l a n t cells [2-5]. P r o t e i n i m p o r t into p l a s t i d s has m a i n l y been studied in vitro with i s o l a t e d p e a c h l o r o p l a s t s (reviewed by D e Boer a n d W e i s b e e k [6]). This imp o r t process is A T P - d e p e n d e n t [7] as is b i n d i n g o f the p r e c u r s o r to the c h l o r o p l a s t e n v e l o p e [8]. A Correspondence to: Douwe de Boer, Agrotechnological Research Institute (ATO-DLO), P.O. Box 17, 6700 AA Wageningen, The Netherlands.
specific s t r o m a l processing p e p t i d a s e (SPP) cleaves off the N - t e r m i n a l transit p e p t i d e when the precursor enters the s t r o m a [9]. S o m e n u c l e a r e n c o d e d p r e c u r s o r proteins, like p l a s t o c y a n i n a n d c o m p o n e n t s o f the oxygen evolving complex, a d d i t i o n a l l y have to cross the thyla k o i d m e m b r a n e system in o r d e r to reach their final location. I m p o r t o f these p r o t e i n s was shown to consist o f two distinct steps a n d the targeting signal o f the p r e c u r s o r has two s e p a r a t e d o m a i n s [10-12]. T h e F i r s t d o m a i n is c o m p a r a b l e to a transit p e p t i d e a n d is cleaved off by the SPP, resulting in an i n t e r m e d i a t e - s i z e d p r o t e i n present in the s t r o m a l fraction. The s e c o n d d o m a i n , the t h y l a k o i d transfer d o m a i n , is essential for thylakold lumen targeting [13] a n d is r e m o v e d by a p r o cessing p e p t i d a s e that is a s s o c i a t e d with the thyla k o i d s [14,15]. To study the t r a n s p o r t o f the t h y l a k o i d lumen p r o t e i n p l a s t o c y a n i n in vivo we t r a n s f o r m e d tobacco p l a n t s with the S i l e n e p r a t e n s i s p r e - p l a s t o -
0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
46
cyanin gene (pPC). The construct was placed under the control of the cauliflower mosaic virus 355 promoter to obtain constitutive expression in all tissues [16]. It was therefore also possible to examine protein transport in non-green tissues. The ability of Silene plastocyanin to take part in photosynthesis was studied in transgenic tobacco plants grown in tissue culture. These plants showed a significantly reduced level of the endogenous tobacco plastocyanin when the heterologous Silene plastocyanin was present. Materials and Methods
Plant transformation and tissue culture The gene for the Silene plastocyanin precursor (pPC) was cloned behind the cauliflower mosaic virus 35S promoter in the binary plant vector pBIN19 [17] as described previously [18]. The construct was conjugated into Agrobacterium tumefaciens LBA4404 containing the modified Tiplasmid pAL4404 [19] in a triparental mating event using the helper plasmid pRK2013 [20]. The construct was introduced into Nicotiana tabacum var. Petit Havanna (SR1) using the leaf disc transformation method [39]. Regenerated plants were selected for kanamycin resistance and grown on tissue culture medium or on soil. The individual transformed plants were numbered A2-1 to A2-8. Plants were grown in tissue culture in small plastic containers with Murashige and Skoog medium without plant hormones [21]. When sucrose was not included in the tissue culture medium, small holes were made in the lid of the containers to optimize diffusion of gases. The lid was subsequently covered with sterile filter paper to prevent infection. To minimize the amount of nutritive matter present in the transferred plants, we only transferred small pieces of stem with one internode to the sucrose-depleted medium. When required, noflurazon was present in the medium at a concentration of 20 ~M. Nucleic acid isolation and blotting D N A was isolated from plants grown on soil using a modified CTAB procedure [22]. Young leaves (about 10 g) were ground in liquid nitrogen and suspended in 10 ml of 2% CTAB, 1.4 M NaC1,
100 m M Tris-HC1 (pH 8.0), 20 m M E D T A and 2% 13-mercaptoethanol. After 30 min incubation at 55°C the suspension was chloroform extracted twice. One tenth volume of 10% CTAB and 0.7 M NaC1 was added before the second extraction. An equal volume of 1% CTAB, 50 m M Tris-HC1 (pH 8.0) and 10 mM E D T A was added and the suspension was incubated for 30 min at room temperature. After centrifugation at 4000 rev./min ( ± 2000 x g) in an SS35 rotor the pellet was washed once with 70% ethanol, resuspended in a CsCI solution and further purified by density centrifugation as described by Maniatis et al. [23]. D N A concentrations were obtained spectrophotometrically assuming that 1 OD260 = 50 t~g DNA/ml. Restriction enzyme digestions, Southern transfer and hybridizations were according to Maniatis et al. [231. R N A was isolated from young leaves of plants grown in soil or in tissue culture by the modified method of Chirwin et al. [24]. Two grams of leaf tissue were ground in liquid nitrogen and 8 ml of 4 M guanidine thiocyanate, 5 m M Na3-Citrate, 0.5% sarkosyl, 0.3% antifoam and 0.1 M 13mercaptoethanol was added. After centrifugation for 10 min in an SS34 rotor at 10 000 rev./min ( + 1 2 000 × g) 1 g/ml CsCi was added to the supernatant. This suspension was transferred to a centrifuge tube on top of a layer of 3 ml 5.7 M CsCI and 0.1 M E D T A (pH 7.5). After centrifugation for 23 h at 29 000 rev./min ( ± 140 000 × g) in an SW41 rotor the pellet was resuspended in 400/A of 10 mM T r i s - H C l (pH 7.5), 5 mM E D T A and 0.!% SDS and extracted twice with butanol/ chloroform (1:4). After ethanol precipitation R N A concentrations were obtained spectrophotometrically assuming that 1 0026o = 40 tzg R N A / ml. R N A electrophoreses and Northern blotting were done using the formaldehyde gel system described by Maniatis et al. [23].
Protein isolation and Western blots Total protein was isolated by grinding tissue in liquid nitrogen and extracting the powder with 30 mM potassium phosphate (pH 7.5), 400 mM NaC1, 2 mM 13-mercaptoethanol, 100 I~M P M S F and 1% Triton X-100. After determining the amount of protein [25], samples of 20 izg protein
47
were electrophoretically separated on a 15% SDS polyacrylamide gel system and blotted as described previously [18].
pBIN 19 RB - - 1
pr.NOS- NPT-t.NOS
~-LACZ I
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.
Chloroplast isolation and fractionation Chloroplasts were isolated from tobacco leaf tissue as described for pea leaves [26]. To remove external proteins the chloroplasts were treated with 0.1 mg/ml thermolysin for 30 min at 4°C. The reaction was stopped by adding EDTA to 10 mM and reisolation of the intact chloroplasts by centrifugation through a 40% Percoll cushion [26]. The chloroplasts were fractionated after they were lysed in 50 mM Hepes (pH 8.0) by spinning down the thylakoids (10 min at 4000 x g). The thylakoids were washed once with 50 mM Hepes (pH 8.0) and 330 mM sorbitol and incubated with 0.4 mg/ml thermolysin for 30 min at 4°C to remove contaminating proteins. The thermolysin treatment was stopped by adding EDTA to 10 mM and reisolation of the thylakoids. Thylakoids were separated in a lumen and a membrane fraction. To achieve this the thylakoids were sonicated after repeated freezing and thawing and the membranes were pelleted by centrifugation for 30 min at 40 000 x g.
Immuno-gold labelling Ultrathin cryosections [27] of tissue of tobacco plants were incubated with antibodies against spinach plastocyanin [28] and labelled with 9 nm protein-A gold particles. Results
Plant transformation and analysis of expression The Silene plastocyanin gene [28] was cloned behind the 35S cauliflower mosaic virus promoter in the binary plant transformation vector pBIN19 [17]; this construct, pBIPC7, is shown in Fig. 1. Transformation of tobacco plants with this construct resulted in eight regenerated kanamycin resistant plants and these were analysed. The presence of the Silene plastocyanin gene in the tobacco genome was determined with a Southern analysis of EcoRI digested chromosomal DNA (Fig. 2a). When the coding sequence of the Silene plastocyanin transit peptide was used as a probe,
plastocyanin transit ~- .............
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1.Skb . . . . . . . . . . . . . .
-~
Fig. 1. The plastocyanin expression plasmid (pBIPC7) used to transform tobacco plants. The construct contains the plastocyanin coding sequence behind the 35S promoter cloned in the EcoRI site of the MCS of the vector pBIN 19 in the orientation indicated. LB and RB denote left and right border of T-DNA, respectively, pr.NOS, nopaline synthetase promoter; t.NOS, nopaline synthetase terminator: NPT, neomycin phosphotransferase gene; MCS, multiple cloning site: c~-LACZ, alpha complementary region of the ~-galactosidase gene: E, EcoR1; H, HindIll.
the expected 1.8 kb EcoRI fragment, containing the 35S-promoter and the plastocyanin coding sequence (see Fig. 1), was detected in the genomic DNA of all transgenic plants. Besides a very faint 1.8-kb band a much larger hybridizing fragment is present in the digested DNA of plant A2-3. The band with the higher molecular weight can be explained when loss of one of the two EcoRI sites in this copy of the integrated DNA is supposed. No cross hybridization with the endogenous tobacco genes or with vector DNA (pBINI9) was seen under the applied stringency (Fig. 2a). The number of integrations into the tobacco genome was estimated by densitometer scanning of the 1.8-kb band using specified amounts of the EcoRI digested plasmid pBIPC7 as a reference. During the calculations a haploid genome size of 1.52 x 109 bp [29] was assumed for tobacco. The number of intact insertions per diploid genome varied from one in A2-3 to eight in A2-4 (see Table I). Transcription of the introduced gene was probed by Northern analysis of leaf RNA. Transcripts were detected in all plants except A2-3 (Fig. 2b).
48
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Fig. 2. Analysis of transgenic plants grown in soil. (A) Southern blot of EcoRl-digested chromosomal DNA. Tobacco (SR1) and Silene chromosomal DNA and vector pBINI9 DNA were used as a control. Ten micrograms of digested DNA is applied per lane. One genome equivalent (a) and five genome equivalents per haploid genome (b) of plasmid pBIPC7 were loaded as references. A Hindlll/Ncol fragment from pPCS74 [10] coding for the transit peptide of plastocyanin was used as a probe. Molecular weight markers are in kbp. (B) Northern blot of total RNA isolated from leaf tissue. RNA isolated from Silene and tobacco (SR I ) leaf tissue was used as a control. Forty micrograms of RNA was applied per lane. Probe and size markers are as described in (A). (C) Western blot of protein isolated from leaf tissue. Protein isolated from leaf tissue of Silene and non-transformed tobacco was used as a control. Twenty micrograms of protein was applied per lane. Antibodies raised against spinach plastocyanin [28] were used to detect plastocyanin. Molecular weight markers are in kDa.
The RNA level in the different plants is not exactly comparable with the number of inserts per individual plant (see Table I), e.g. A2-5 has a higher RNA level than A2-4 although the number of inserts in the genome is lower. Position effects may well be the cause of these differences. The size of the transcripts in all plants was comparable to the size of the plastocyanin transcript in Silene plants (about 780 bp). This would be expected when normal transcription termination and polyadenylation of the introduced gene occurs in the transgenic tobacco plants. Cross hybridization with endogenous plastocyanin transcripts was not observed under these conditions. Translation of the transcripts in tobacco leaf tissue of plants grown in soil was analysed in protein blotting experiments using antibodies directed against spinach plastocyanin (Fig. 2c). Control tobacco plants show two endogenous plastocyanin
bands, which both react with the plastocyanin antibody. Six out of eight plants tested show, besides the two endogenous plastocyanin bands, a new protein band that has the same mobility as the Table 1.
Genomic copy numbers and RNA expression levels of the Silene plastocyanin construct in the transgenic plants. Plant no.
Estimated copy no. per diploid genome
Relative amount of RNA (%)~
A2-1 A2-2 A2-3 A2-4 A2-5 A2-6 A2-7 A2-8
6 2 1 8 5 2 2 4
31.1 17.4 0.0 48.4 100.0 11.1 48.4 28.5
aThe RNA levels are given as percentages of the amount found in A2
49
mature Silene protein. Although we did not quantify the Western blot it is apparent that there is a correlation between the transcript level of the Silene gene and the amount of Silene plastocyanin detected. Plant A2-6 expresses the protein at a low level; overloading the gel is necessary to visualize the plastocyanin band. This plant also has a low level of transcription. The Silene protein could not be detected in plant A2-3 as could be expected from the Northern blotting experiments. There is apparently no effect of the presence of the Silene plastocyanin on the level of the endogenous tobacco plastocyanin in these plants, grown in soil. All eight plants were morphologically equal to wild type (SR 1) plants. In the non-green petal tissue of flowers, endogenous tobacco plastocyanin is not expressed. However, the Silene plastocyanin protein was shown to be present in petal tissue when assayed with protein blotting experiments (Fig. 3). All transformed plants with expression in leaf tissue also show expression in petal tissue. The same holds for root tissue, although the amount of Silene protein is low as compared to petal tissue (not shown). Cellular localization of Silene plastocyanin The Silene plastocyanin protein synthesized in leaf tissue of transgenic tobacco plants migrates with the same mobility and therefore has the apparent molecular weight of mature plastocyanin isolated from Silene leaves. The Silene precursor protein is apparently processed to its mature size. The localization of the Silene protein in the tobacco leaf cell was determined by isolation and fractionation of chloroplasts of the transgenic plant A2-5. The Western blot of the various fractions shows that both Silene and tobacco plastocyanin behave identically during fractionation (Fig. 4). Both proteins are protected against protease treatment of the intact chloroplast or the intact thylakoids and fractionate only in the thylakoid lumen fraction. Electron microscopical analysis of immuno-gold labelled coupes of leaf tissue also localizes all plastocyanin exclusively to the thylakoids (not shown). The Silene protein in petal and root tissue has the same electrophoretic mobility as mature plastocyanin in leaf tissue (see Fig. 3). The protein
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is therefore expected to be transported into the plastids present in these tissues. Electron microscopy in combination with immuno-gold labelling shows that in petal tissue plastocyanin is indeed present inside the chromoplasts (not shown). Only a limited amount of background staining is observed outside the plastids. Expression of plastocyanin in plants grown in tissue culture In transgenic plants analysed after growth in soil, no changes in the level of endogenous plastocyanin was observed (Fig. 2c). However, with plants that were permanently grown in tissue culture the amount of endogenous plastocyanin was found to be reduced significantly, whereas the amount of the Silene protein in most plants was unchanged. Figure 5a shows a Western blot of total protein isolated from such plants. A2-1 and A2-8, two plants that express the Silene plastocyanin gene at about the same level in tissue culture as when grown in soil (Fig. 2c), show a striking change in the level of endogenous tobacco plastocyanin. The amount of it was reduced by at least a factor ten. A faint signal of endogenous plastocyanin could be detected after overloading the gel. The quantity of other chloroplast proteins was not reduced, as was checked by Coomassie blue staining of total leaf protein after elec-
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Fig. 4. Localization of Silene plastocyanin in chloroplasts of the transgenic tobacco plant A2-5. (A) Western blot of total proteins isolated from chloroplasts. Protein isolated from Silene and non-transformed tobacco (SRI) leaves was used as a control. PC antibodies as indicated in Fig. 2c. 1, leaf protein; c, chloroplast protein. Arrows point at the position of mature Silene plastocyanin. (B) Western blot of total protein isolated from fractionated chloroplasts. 1, leaf protein; t, total thylakoid protein; tl, thylakoid lumen protein; tm, thylakoid membrane protein. Antibodies, controls and arrows as in (A).
trophoresis on SDS-PAGE (not shown). This absence of tobacco plastocyanin in tissue culture could be a consequence of the growth condition or of the presence of the Silene protein. To test this a non-transformed tobacco plant and the transgenic plants A3-3 and A3-6, which were transformed with a different gene cloned in the same vector, were also analysed. The reduction of the endogenous plastocyanin was not observed with these plants when grown in tissue culture (Fig. 5a). Therefore the effect, although only observed in
plants grown in tissue culture, is correlated with the presence of the Silene protein. This strong reduction of tobacco plastocyanin raises the interesting question whether the Silene plastocyanin can functionally replace the tobacco plastocyanin in photosynthesis. However, this question could not directly be answered, although these plants were able to grow in tissue culture medium in the absence of endogenous plastocyanin. Due to the presence of sucrose in the tissue culture medium these plants did not need to photosynthesize in order to grow. This was clearly shown when we included in the medium 20 ~zM norflurazon, an inhibitor of carotenoid biosynthesis, which leads to photo bleaching in the light [30]. Although the plants that were grown in this medium were white, they were able to grow heterotrophically (Fig. 6). Photosynthetically active plants growing in tissue culture were obtained by the exclusion of sucrose from the medium. Both plants A2-1 and A2-8 and the control plants were able to grow on sucrose-depleted medium. They both grew with a reduced growth rate and they were comparable to the green plants grown on a medium with sucrose (Fig. 6). Inclusion of 20 #M norflurazon in the medium without sucrose
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Fig. 5. Western blot of total protein isolated from plants grown in tissue culture. (A) Total protein isolated from leaf tissue of plants grown on tissue culture medium with sucrose. (B) As (A) but plants were grown on tissue culture medium without sucrose. For use as controls, protein was isolated from non-transformed tobacco (SR1) and two tobacco plants (A3-5 and A3-6), that were transformed with an arbitrary pBINI9 construct. These control plants were also grown in tissue culture• Protein isolated from Silene leaf tissue was used as a reference for mature Silene plastocyanin. Antibodies were as indicated in Fig. 2c. Molecular weight markers are in kDa. Arrows point at the position of mature plastocyanin.
51
Fig. 6. Tobacco plants grown in tissue culture media of different composition. The green (dark) plant at the left was grown on normal tissue culture medium with sucrose. The white plant on the right was grown on tissue culture medium with sucrose and 20 ~M norflurazon. The small white plants in the middle were grown on tissue culture medium without sucrose in the presence of 20/~M norflurazon.
resulted in very small white plants which died after a few weeks (Fig. 6). This convincingly showed that photosynthetic activity was necessary on a medium without sucrose. Reduced levels o f endogenous plastocyanin were found in tobacco plants expressing Silene plastocyanin, likewise whether they were grown in tissue culture medium with sucrose or without sucrose (Fig. 5b). A higher level o f endogenous plastocyanin would have been expected in case o f an inactive Silene plastocyanin protein. Therefore, we conclude that Silene plastocyanin is able to take part in photosynthesis in these transgenic tobacco plants. We can not exclude the possibility that the residual endogenous plastocyanin is taking part in photosynthesis, but we consider it unlikely that this small a m o u n t is sufficient for growth on a medium without sucrose.
Discussion We have obtained transgenic t o b a c c o plants
that properly express the Silene plastocyanin gene. The Silene plastocyanin protein could be separated from the two endogenous ones because o f differences in electrophoretic mobility on SDSP A G E . Our gelelectrophoresis system revealed the presence o f two endogenous plastocyanin bands in tobacco cells. Last and G r a y [31] only detected one band with a non-denaturing gelelectrophoresis system. The presence o f two different plastocyanin proteins is, however, very likely considering the allo-tetraploid nature o f N. tabacum [32]. Expression of the Silene plastocyanin protein did not affect the level o f endogenous plastocyanin when the plants were grown in soil. Similar results were also found in other studies in which foreign plastocyanin proteins were expressed in transgenic plants [18,31]. The Silene protein is imported into chloroplasts and routed towards the thylakoid lumen, where it is processed to its mature size. This means that the transit peptide o f the Silene protein is fully recognized by the tobacco protein transport ma-
52 chinery. Similar results were obtained with tomato plants transformed with the Silene plastocyanin gene [18]. Therefore it is likely that the mechanism of chloroplast protein import and routing is conserved among higher plants. Small differences will probably only affect the efficiency of the transport process. This view is also supported by in vitro import experiments with isolated pea chloroplasts. These pea chloroplasts are able to import proteins from a wide range of different plants (for an overview see Ref. 16). Due to the constitutively expressed CaMV 35S promoter Silene plastocyanin is also synthesized in petal and root tissue of the six different transgenic plants that were analysed. The endogenous plastocyanin is not made in non-green tissue. Silene plastocyanin is localized inside the plastids in these tissues, even though they are morphologically distinct from chloroplasts. Similar observations were made when Silene plastocyanin was expressed in various tissues of transgenic tomato plants [18]. Non-green plastids were also found to be competent for import of chloroplast proteins in in vitro import experiments [33-35]. Also amyloplast precursor proteins can be imported into chloroplasts in vitro [36]. In both tomato and tobacco the Silene plastocyanin protein was found processed to its mature size in these non-green tissues, although a real thylakoid membrane system is not present in the organelles. Experiments in which the precursor of plastocyanin was imported into castor bean leucoplasts in vitro showed the presence of an intermediate sized protein in the organelle [35]. This indicates that in vivo the presence of a mature-sized protein might reflect the situation after an additional aspecific processing. The mature part of the protein is probably more resistent to protease when folded properly after uptake of Cu >. Uptake of Cu 2+ can occur whenever mature plastocyanin is formed. Apoplastocyanin is shown to take up Cu 2+ in vitro without the help of a protein factor [37], and plastocyanin also incorporates Cu 2+ when it is mistargeted to the stroma in in vitro import experiments [38]. Therefore the mature part of the protein is expected to be selectively stabilized. This shows that it might be wrong to conclude that a protein is imported into organelles only by the observation that the precursor is processed.
We now can extend our previous conclusions [18] to the more general statement that the protein import machineries of different plastids in plants do not discriminate between proteins destined for either of them. Differentiation of the plastids during development seems to have no influence on the mechanism of protein import. If all different plastids are indeed capable of importing all the proteins present in either of them, it is difficult to believe that they possess a wide range of specific receptor proteins, one for each protein imported. For this reason it is more likely that there is only one receptor, or at most a few, with low specificity. In contrast to plants grown in soil, transgenic tobacco plants grown in tissue culture show a highly reduced level of endogenous plastocyanin when the Silene protein is present. This decrease in endogenous plastocyanin made the Silene protein the prevailing plastocyanin in these plants. This made it possible to study whether a foreign plastocyanin protein is able to take over electron transfer in the thylakoid lumen in vivo. During growth conditions that required photosynthesis we observed growth of both transgenic and control tobacco plants. The transgenic plants still only expressed Silene plastocyanin under these conditions, suggesting that this plastocyanin is active in photosynthesis in vivo. It is not completely excluded that the very low amount of endogenous plastocyanin, present in these plants accounts for all the photosynthetic activity. However, we consider this unlikely since no obvious differences were observed between control plants, which expressed tobacco plastocyanin at normal levels, and the transgenic plants, which only contained trace amounts of endogenous plastocyanin. We do not know why the endogenous plastocyanin protein is down-regulated in plants grown in tissue culture and not in plants grown in soil. It is possible that the presence of the Silene protein down regulates the endogenous plastocyanin level, but this down regulation is not sufficient when there is a strong demand for the protein, e.g. during rapid growth on soil. Measurements on pea plastocyanin isolated from transgenic tobacco plants showed that this foreign plastocyanin protein was correctly folded and able of electron transfer in vitro [31]. Taken together we can conclude that a plastocyanin gene
53 that duce tein that
is e x p r e s s e d in a f o r e i g n p l a n t is a b l e t o a functional protein capable of proper transport towards the thylakoid lumen t h i s p r o t e i n is a l s o a b l e t o p a r t i c i p a t e in
proproand elec-
11
tron transfer during photosynthesis. This proper f u n c t i o n i n g is e s s e n t i a l f o r in v i v o s t u d i e s w i t h a u t h e n t i c o r m u t a n t p r o t e i n s in t r a n s g e n i c p l a n t s .
12
Acknowledgements We thank Dr. Sjef Smeekens for his participation and Mr. Harry Bettenbroek and Lucy Molen-
13
dijk for technical assistance during of this work. We are grateful to SANDOZ A.G. in Basel for his azon. This work was supported
14
Netherlands Organization Pure Science (SON/ZWO).
the initial Parts D r . J. H a r r o f gift o f n o r f l u r in P a r t b y t h e
for Advancement
of 15
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