Nuclear-organelle interactions: Nuclear antisense gene inhibits ribulose bisphosphate carboxylase enzyme levels in transformed tobacco plants

Cell, Vol. 55,

673-681,

November 18, 1988, Copyright 0 1988 by Cell Press

Nuclear-Organelle Interactions: Nuclear Antisense Gene Inhibits Ribulose Bisphosphate Carboxyiase Enzyme Levels in Transformed Tobacco Plants Steven R. Rodermel: Marilyn S. Abbott,t Lawrence Bogorad’ + The Biological Laboratories Harvard University 16 Divinity Avenue Cambridge, Massachusetts 02138 tAnheuser Busch Company Corporate Research and Development 1101 Wyoming St. Louis, Missouri 63118

and

Summary The biosynthesis of ribulose bisphosphate carboxylase (RUBISCO) provides a model system for studying the coordination of nuclear and organelle gene expression, since this abundantly transcribed and expressed chloroplast enzyme is composed of small (SS) and large subunits (LS) encoded by a nuclear multigene family and a single chloroplast gene, respectively. We have tested the possibility that SS mRNA or protein levels affect LS mRNA amounts or LS protein production and accumulation. We find that expression of antisense DNA sequences for the SS in transgenic tobacco plants drastically reduces the accumulation of SS mRNA and SS protein. These changes are accompanied by corresponding reductions of LS protein but not LS mRNA amounts; accumulation of the LS protein appears to be regulated by translational and posttranslational factors. We also find that the transgenic plants display striking variations in growth that are correlated with antisense gene dosage. Introduction Many, and perhaps all, multimeric protein complexes within chloroplasts and mitochondria are composed of subunits synthesized by two separate genetic systemsthat of the nucleus and the organelle (Bogorad, 1982). Despite the vast difference in copy number between the nuclear and organellar genomes, free subunits of these complexes do not accumulate to an appreciable extent. This suggests that regulatory mechanisms exist to coordinate subunit synthesis in the two compartments. Ribulose-15bisphosphate carboxylase/oxygenase (E. C. 4.1.1.39), or RUBISCO, is a multimeric protein complex present in the chloroplasts of higher green plants and photosynthetic microorganisms. It is the most abundant soluble plant protein (Ellis, 1979) and is localized in the chloroplast stroma where it catalyzes the first step in both the COP fixation and photorespiratory pathways (reviewed in Miziorko and Lorimer, 1983). The holoenzyme is composed of eight catalytic large subunits (LS; M, of approximately 55 kd each) and eight small subunits (SS; M, of approximately 14 kd each; reviewed in Miziorko and

Lorimer, 1983). The SS are encoded by small nuclear multigene (rbcS) families, translated on free cytoplasmic ribosomes as precursors, and then transported into the chloroplast posttranslationally (reviewed in Schmidt and Mishkind, 1986; Kuhlemeier et al., 1987). The LS are encoded by a single gene @CL) on the chloroplast chromosome (Coen et al., 1977; Bedbrook et al., 1979) and translated on membrane-bound and free stromal ribosomes (Hattori and Margulies, 1986). Newly synthesized LS monomers associate with two kinds of nuclear-encoded large subunit binding proteins (LSBPs) prior to their association with SS monomers to form the holoenzyme (Barraclough and Ellis, 1980; Roy et al., 1982; Cannon et al., 1986; Hemmingsen and Ellis, 1986; Musgrove et al., 1987). The levels of rbcS and &CL transcripts in higher plants and some algae are regulated by light, as well as by celland tissue-specific factors (reviewed in Kuhlemeier et al., 1987). The effects of light on rbcS mRNA appear to be mediated by the phytochrome pigment at the level of transcription initiation (Tobin and Silverthorne, 1985). In some plants, increases in the transcript pools from both genes during the course of normal light-induced development, or during the greening of etiolated tissues, are accompanied by corresponding increases in the accumulation of RUBISCO, indicating that accumulation of the holoenzyme is regulated primarily at the level of transcript pools (reviewed in Tobin and Silverthorne, 1985). In other plant systems, however, coordinate changes in subunit accumulation are not closely coupled with changes in the amounts of one or both transcripts, suggesting that posttranscriptional controls may be superimposed upon transcription coordinating mechanisms to fine-tune the levels of the LS and SS (e.g., lnamine et al., 1985; Kirk and Kirk, 1985; Berry et al., 1985, 1986; Steinbiss and Zetsche, 1986; Abbott and Bogorad, 1987; Nikolau and Klessig, 1987; Radetzky and Zetsche, 1987; Sasaki et al., 1987; Sheen and Bogorad, 1986). The question arises as to whether some degree of coordination of rbcS and fbcL transcript and protein levels is mediated via signals perceived independently in the two compartments or via a primary signal perceived in one compartment and transduced by a secondary signal to the other. For example, if there is a single primary perceptive system localized in the nuclear-cytoplasmic compartment affecting expression in both compartments, could the second messenger be the pre-SS or SS protein? Or, could a different second messenger be generated in response to the level of rbcS transcripts or SS proteins? One approach to determining whether rbcS transcripts or their translation products either directly or indirectly influence &CL transcription and translation is to perturb the levels of rbcS transcripts and/or SS proteins and then determine the effects of such alterations upon the rbcL transcript and LS protein levels. Inhibitors of transcription or translation in the nuclear-cytoplasmic compartment have been used previously for this purpose (e.g., Sasaki. 1986; Radetzky

Cell 674

pAC1352

3

72325c

T&i‘v

.

rbcS

5’.35s

3’.35s -

Figure I. Construction Vector

5

of the r&S

Antisense

mRNA

100 bp

Expression

A 321 bp Xmnl-EcoRI &&containing subfragment was isolated from pSEM1 (Pinck et al., 1964) the ends of the fragment were filled in with Klenow DNA polymerase, and the fragment was subcloned into the Smal site of pGEM1 (Promega Biotec) to produce pGEM-SS. Following conversion of the Sac1 site in the polylinker region of pGEM-SS into a Bglll site, a 340 bp BamHI-Bglll subfragment (containing all of the r&S sequences present in the original Xmnl-EcoRI subfragment) was isolated and introduced into the Bglll site of pAC1352 (A. Cheung and L. Bogorad, unpublished data) to generate pTASS. pAC1352 is a CaMV 3% promoter/terminator cassette in which the Bglll site separates the 3% TATA region (“TATATAA”) and polyadenylation sequences (“AATAAA’) (Guilley et al., 1962; Odell et al., 1965). pAC1352 also includes a subfragment of pLGVneotlO3 (Hain et al., 1965) that contains the nopaline synthetase promoter (5’~NOS) fused to the coding region of the neomycin phosphotransferase gene of Tn5 (NPT//) and the polyadenylation signal of the octopine synthase gene (3’-OCS). The nos and 355 promoter regions are oriented in pACl352 such that transcription is divergent. (B = BamHI; Bg = Bglll; P = Pstl; RB and LB, right and left borders of the T-DNA, respectively.)

and Zetsche, 1987). However, the pleiotropic effects of such agents do not permit definitive conclusions. To avoid such difficulties, we have used antisense RNA technology to produce mutant plants in which SS metabolism is perturbed. In this paper, we demonstrate that mutants with sharply depressed levels of r&S mRNA and SS protein can be generated by the introduction of rbcS antisense DNA sequences into the nuclear genome of tobacco. These mutants also have reduced levels of RUBISCO, but normal amounts of &CL mRNA are maintained. These effects are striking since rbcS transcripts are among the most plentiful leaf mRNA species of nuclear origin (Tobin and Silverthorne, 1985). Therefore, the use of antisense genes to manipulate the expression of endogenous plant genes seems promising for studies of plant development and genetic engineering.

Antisense Vector Construction and Agrobacterium-Mediated lkansformation of Tobacco The construction of an rbcS antisense vector (pTASS) for use in Agrobacterium-mediated transformation experiments is outlined in Figure 1. The rbcS DNA fragment present in this vector was derived from pSEM1, a Nico-

Figure 2. Copy Number of rbcS Antisense Plants

Genes in the Transgenic

Southern hybridization experiments were performed with 10 pg of Bglll-digested genomic DNA from transformants 3,5,7,23, and 25 and from an SRI control plant (“C). The filters were probed with the 1.6 kbp Pstl subfragment of the NPTll gene from pTASS (see Figure 1). Approximate molecular lengths (in kbp) are indicated on the left,

tiana sylvestris rbcS cDNA clone (Pinck et al., 1984), and includes sequences extending from 22 bp upstream of to 300 bp downstream of the initiation codon of the gene. These sequences are 99.7% homologous to sequences in two genomic rbcS genes from Nicotiana tabacum that account for the bulk of rbcS mRNA production in the leaves of this species (Mazur and Chui, 1985; C’Neal et al., 1987). The pSEM1 rbcS fragment was cloned into a cauliflower mosaic virus (CaMV) 35s promoter/terminator cassette (pAC1352) in such an orientation that transcription from the high expression level 35s promoter (e.g., Bevan et al., 1985; Sanders et al., 1987) would result in the production of rbcS antisense mRNAs in transgenic plants. A DNA fragment containing a copy of the NPTll gene fused to the nopaline synthase promoter and octopine synthase terminator elements was included in this vector as a selectable marker. pTASS was introduced into the nuclear genome of tobacco SRl plants by Agrobacterium-mediated DNA transfer methods (as described in Experimental Procedures), and five kanamycin-resistant plants were selected for further study (transformants 3, 5, 7, 23, and 25). Copy Number of Foreign DNA Sequences in the lkansgenic Plants To confirm the presence of rbcS antisense DNA sequences in the chromosomes of putative transformants 3, 5, 7, 23, and 25, Southern hybridization experiments (Southern, 1975) were performed with restriction digests of DNA from each transformant using an NPTII-containing subfragment of pTASS as a probe. The DNAs were digested with either BamHl or Bglll, each of which recognizes a single site between the T-DNA border sequences of pTASS (see Figure 1). It would thus be anticipated that for each copy of the foreign pTASS sequence present in the genome of these plants, digestion with either of these enzymes would yield a uniquely sized chimaeric restriction fragment containing sequences complementary to the NPTll probe. The Southern hybridization experiments showed that Bglll or BamHl digests of the DNAs from

Antisense 675

mRNA Inhibition

of RUBISCO

SR1 C

E

Levels

a-SENSE

Table 1. rbcS a d rbcL mRNA and SS and LS Protein Levels in the Transformed Versus Control Plants

3723255

RNA (% Controls)

LS-

ss-

Figure 3. Levels of the rbcS and rbcL mRNAs Control Plants

in Transformed

and

Northern hybridization experiments were performed with 10 ug of RNA from transformants 3, 5, 7, 23, and 25 and from two control plants (“C and “E”). The filters were probed with pSEM1 (Pinck et al., 1984) to detect rbcS mRNAs (“Ss”) or pTB5 (Shinozaki and Sugiura, 1982) to detect rbcL mRNAs (“Ls”).

transformants 3, 7, 23, and 25 each contain a single NPTll sequence, whereas transformant 5 appears to contain several hybridizing bands (Figure 2). This suggests that all of the transformed plants contain at least one copy of the r6cS antisense gene, but that transformant 5 may contain four or more copies of this gene. While the multiple bands of transformant 5 differ in intensity, these bands assort independently in the F, progeny of this transformant (see Figure 8 and subsequent discussion). It is therefore likely that these bands represent integration events at several sites involving all or part of pTASS. The Accumulation of the Large and Small Subunit mRNAs Can Be Uncoupled To assay the steady state levels of r&S and &CL transcripts in the transformed and control plants, Northern hybridization experiments were performed with samples of RNA isolated from plants growing on tissue culture medium containing sucrose. The filters were probed with pTB5 DNA specific for the N. tabacum &CL gene (Shinozaki and Sugiura, 1982) or with the N. sylvestris rbcS cDNA clone (pSEM1; Pinck et al., 1984) used in the construction of pTASS. The latter probe is complementary to all members of the rbcS gene family in N. tabacum @ ‘Neal et al., 1987). A typical Northern hybridization experiment (Figure 3) shows that the &CL transcript levels are similar in the transformed and control plants but that the rbcS mRNA levels are depressed to varying degrees in the transformed plants. Densitometric analyses of replicate samples (summarized in Table 1) indicate that the levels of rbcL mRNA in the transformed plants do not differ significantly from the controls. In contrast, the rbcS mRNA levels are depressed approximately 4-fold in transformants 3, 7, 23, and 25 and approximately lo-fold in transformant 5. Therefore, the accumulation of rbcS and r&L transcripts is not tightly coupled in this system. Identification of the rbcS Antisense Transcript On overexposed autoradiographs of Northern filters probed with double-stranded rbc&specific DNA, a low-

Protein (% Controls)

Transformants

rbcS

rbcL

ss

LS

3 5 7 23 25

33 12 25 26 26

79 101 100 88 96

53 37 81 63 49

55 38 65 71 60

-

Densitometric scans of autoradiographs or Coomassie blue-stained gels were made and the areas under the peaks were measured. To assure linearity, all gels contained a dilution series of one sample, and autoradiographs were exposed for varying amounts of time. Each value represents the mean from replicate experiments for each transformant expressed as a percentage of the control plant mean. In each experiment, the variation in the individual transformed plants was within the range of variation observed among the control plants (from about 20%-27%). The values obtained from autoradiographs in the Northern experiments (“RNA” column) are derived from up to eight separate hybridizations performed with a total of three different RNA preparations; up to three control plants were used in each experiment. The estimates of protein amounts (“Protein” column) are derived from three separate protein preparations.

abundance 0.75 kb RNA species is visible in lanes containing samples of RNA from the transformed plants (e.g., Figure 4, lane 1). To investigate whether this RNA species is a product of the rbcS antisense gene, strand-specific probes were constructed by subcloning the rbcS cDNA subfragment from pSEM1 into an SP6iT7 expression vector, and radiolabeled “sense”and “antisense”rbcS mRNAs were synthesized. These RNA probes were hybridized to Northern filters containing RNAs from transformant 5 and a control plant. When the filters were probed with labeled “sense” RNAs, hybridization to the 0.75 kb transcript was observed only in the RNA sample from transformant 5 (Figure 4, lane 2). Conversely, when the filters were

ssa-SS12 Figure 4. Identification formed Plants

of the rbcS Antisense

3 Transcript

in Trans-

Lane 1: nick-translated pSEM1 was hybridized to a Northern filter containing 10 ug of RNA from transformant 5. Lanes 2 and 3: the 570 bp rbcS cDNA fragment from pSEM1 (Pinck et al., 1984) was subcloned into the Pstl site of 16176 (IBI), and radiolabeled “sense” or ‘antisense” RNAs were synthesized in vitro using either SP6 polymerase (to make “sense” RNAs) or T7 polymerase (to make “antisense” RNAs) (Promega Biotec). The labeled RNAs were hy bridized to Northern filters containing 10 ug of RNA from transformant 5 and a control plant (“C’). Lane 2 shows the results of hybridization with the “sense” RNA probe, and lane 3 shows the results of hybridization with the “antisense” RNA probe. “Ss” = the 0.95 kb rbcS mRNAs; “A-W = the 0.75 kb rbcS antisense mRNAs.

Cell 676

C

E

3

723255

C

E

3

723255

LS-

ss-

Figure 6. In Vivo Protein Synthesis Plants Figure 5. Protein Accumulation

in the Transformed and Control Plants

Twenty-five micrograms of soluble protein from the transformed (3, 5, 7, 23, and 25) and control plants (C and E) were electrophoresed through a discontinuous SDS-polyacrylamide gel and stained with Coomassie blue. The LS and SS bands were identified by Western analysis using antibodies generated against the tobacco LS and SS proteins.

probed with labeled ‘antisense” RNAs, hybridization was observed only to rbcS mRNAs in both samples (Figure 4, lane 3). Assuming that CaMV 35s promoter and polyadenlyation signals are used, a 0.75 kb RNA would fall within the expected size range of a transcript encoded by the inserted rbcS antisense gene. We conclude that the low abundance 0.75 kb mRNA detected in the transgenic plants is the rbcS antisense transcript. Coupled with the results in Figure 3, the data are thus consistent with the hypothesis that antisense mRNA exerts its inhibitory effect on rbcS mRNA levels by forming sense:antisense RNA duplexes, which are subsequently degraded, e.g., at the level of processing, transport or translation, although the data do not rule out the possibility that the antisense RNAs bind to DNA and block rbcS transcription. The detailed mechanism of antisense mRNA inhibition in this situation remains to be ascertained. RUBISCO Accumulation Is Coupled to Transcriptional and Posttranscriptional Events The data in Figure 3 demonstrate that reductions in rbcS mRNA levels have little, if any, influence on rbcL mRNA levels. To test whether the lowered rbcS mRNA levels affect the accumulation of the LS or SS proteins, soluble proteins extracted from the leaves of transformed and control plants growing on sugar-containing tissue culture medium were compared by SDSpolyacrylamide gel electrophoresis (Figure 5); equal amounts of protein were applied to each lane of the gel. Densitometric analyses of replicate samples of stained gels (Table 1) reveal that both the LS and SS levels in transformants 3, 7, 23, and 25 are decreased by approximately 40% as compared with the control plants and that the amounts of both proteins in transformant 5 are depressed about 60% in comparison with the controls. Similar results were obtained in Western blot analyses (Towbin et al., 1979) using antibodies gener-

in the Transformed

and Control

Equal cpm of soluble protein from transformed (3, 5, 7, 23, and 25) and control plants (C and E) labeled in vivo fpr 1 hr with sY%methionine were electrophoresed through a discontinuous SDS-polyacrylamide gel and fluorographed. The LS and SS bands were identified by Western blot analyses, as described in the legend to Figure 5. The two heavily labeled bands migrating between the LS and SS in the control lanes are immunologically related to the LS. The intensities of these bands were generally related stoichiometrically to the intensity of the LS band in the transformed and control plants; it is not understood why they are more prominent in the control plants in this particular experiment.

ated against the N. tabacum LS and SS proteins (data not shown). Coordinate reductions in LS and SS protein levels even more pronounced than those in Figure 5 were obsewed when the amounts of protein loaded onto the gel were normalized to equal amounts of chlorophyll (as a measure of membrane protein) rather than equal amounts of soluble protein (data not shown). This is a reflection of the fact that the amount of soluble protein per unit of chlorophyll is lower in transformed than in the control plants (averaging from 55%-90% of the control values, depending upon the transformant). The coordinate reductions in amounts of the LS and SS as fractions of total soluble protein indicate that the normal stoichiometry in the steady state pool sizes of the two subunits is maintained in the transformed plants. Furthermore, electrophoresis of the protein samples under nondenaturing conditions indicated that the levels of RUBISCO holoenzyme are as depressed as the individual subunit pool levels in each of the transformants (data not shown). Thus, the accumulation of the holoenzyme appears to be related to the accumulation of rbcS mRNAs, although this relationship is not strictly proportional since SS protein levels in the transformants are less severely reduced than rbcS transcript levels. In contrast, the accumulation of RUBISCO is not directly related to rbcL mRNA levels, suggesting that some as yet unidentified posttranscriptional events play a major role in the accumulation of the LS protein. To test whether translation might be the posttranscriptional event that regulates the accumulation of the LS protein, two leaves from each of the transformed and control plants were supplied with SS-methionine for 1 hr before harvest. With the exception of transformant 5, the data in Figure 6 indicate that the amount of 3% incorporated into

$lsense

mRNA inhibition

of RUSISCO

Levels

LS is approximately equal in the transformed and control plants. This result, coupled with the observation that these transformants have normal amounts of r&L mRNA (Figure 3), is consistent with the hypothesis that &CL mRNAs are translated with similar efficiencies in the transformed and control plants, but that the LS protein is degraded more rapidly in the transformants, perhaps due to a lack of sufficient SS. However, it is possible that translation may also be inhibited when the amount of rbcS transcripts is severely reduced, since LS synthesis is markedly depressed in transformant 5 (Figure 6) which also has a normal level of rbcL mRNA (Figure 3). Alternatively, although all the other transgenic plants in these experiments incorporated approximately equal amounts of 35S-methionine into LS during the labeling period, it is possible that the reductions in LS labeling in transformant 5 may be due to enhanced LS degradation. Additional

studies of plants with very low levels of rbcS mRNA are required to address this question. inhibition of Growth Is Correlated with rbcS Antisense Gene Dosage Transformant 5 contains the largest number of pTASS DNA sequences in its genome, and the levels of rbcS mRNA and RUBISCO holoenzyme are the most severely depressed in this transformant. Various F, progeny from the self-fertilization of transformant 5 exhibit markedly different rates of growth (Figure 7). Southern hybridization experiments performed with Bglll-digested DNA from these plants (Figure 6) show that there is a negative correlation between growth rate and the apparent copy number of foreign pTASS DNA sequences in the genomes of these plants, i.e., the tiny (‘T’) plant has the highest number of copies, followed by the small (‘9) and then the medium-

P

FI BMS

Figure 8. Segregation Transformant 5

of pTASS Sequences

T

among the F, Progeny of

The Southern experiments were performed as described in the legend to Figure 2 using Bglll-digested DNA from each of the Fr progeny plants and the control plant shown in Figure 7. DNA was also isolated from the parental (“P”) transformant 5 plant that served as the source of the F, progeny (see Figure 2). Approximate molecular lengths (in kbp) are indicated on the left.

sized (“M”) plants; the big (“B”) plant appears to be a wildtype segregant since it contains no pTASS sequences. The intensity of hybridization to the 3.4 kbp band in the “tiny” plant is striking; perhaps pTASS sequences have been amplified in this plant by some unknown mechanism. Examination of other progeny from transformant 5 has yielded results similar to those in Figures 7 and 8. This suggests that in this system, over some as yet undetermined range, the greater the expression and dosage of antisense genes, the slower the plants grow. Although preliminary experiments have shown that the growth rates of the antisense progeny plants are positively correlated with rbcS mRNA pool sizes and RUBISCO levels, quantitative relationships between growth rates and RUBISCO levels remain to be determined. Discussion The accumulation of subunits of multimeric protein complexes within the chloroplast or mitochondrion requires the coordination of biosynthetic activities between the organelle and the nucleus. In the case of RUBISCO, the first level of coordination during development appears to be in the abundance of rbcS and rbcL transcripts (e.g., Tobin and Silverthorne, 1985; Nikolau and Klessig, 1987; Sasaki et al., 1987; Sheen and Bogorad, 1986). The regulation of these transcript pool sizes may be a consequence of information flow from one compartment to the other, or alternatively, of signals operating independently in the two compartments. We have tested whether rbcL transcript and protein levels are regulated by the amounts of rbcS mRNA and protein in the cell by using antisense RNA technology to generate mutants of tobacco with depressed rbcS mRNA and SS protein levels. Our observation that the accumulation of rbcL mRNAs is unaffected in these mutants is consistent with the results of short-term inhibitor studies suggesting that rbcL mRNA accumulation is generally independent of nuclear transcription (Sasaki, 1986; Radetzky and Zetsche, 1987). However, since inhibitors of

transcription and translation have broad effects, it is not possible to determine whether the results from such experiments are due to the elimination of independent signals, of signals generated in response to rbcS transcript or SS protein levels, or of the rbcS mRNA and SS molecules themselves. Because of the specific perturbation achieved in the present study, it is clear that if a signal is transduced from the nuclear-cytoplasmic compartment to the chloroplast to regulate rbcL mRNA amounts, it is not rbcS mRNA or protein or second messengers generated in response to the levels of these molecules. The coordinate reductions in the accumulation of LS and SS protein appear to be loosely coupled to the reduced rbcS mRNA levels in the antisense mutants. This argues that the abundance of rbcS mRNA may be the major factor defining RUBISCO levels, but that other mechanisms, e.g., LS degradation, are responsible for finetuning the amounts of these proteins in the cell. In general, these findings are in accordance with previous data showing that LS and/or SS accumulation can be influenced by translational factors (e.g., lnamine et al., 1985; Kirk and Kirk, 1985; Berry et al., 1985, 1988; Abbott and Bogorad, 1967; Nikolau and Klessig, 1987; Radetzky and Zetsche, 1987; Sheen and Bogorad, 1986) and that excess amounts of the LS and/or SS proteins can be degraded posttransiationally within the chloroplast (e.g., Roy et al., 1982; Schmidt and Mishkind, 1983; Hildebrandt et al., 1984; Spreitzer et al., 1985; Cannon et al., 1986; Radetzsky and Zetsche, 1987). However, our observations further suggest that translational restrictions on LS accumulation may come into play only when rbcS mRNA or SS protein pools are very small (e.g., transformant 5). This indicates that a hierarchy of posttranscriptional controls may be involved in regulating the amounts of the LS protein. Experiments are in progress to test this hypothesis. Previous investigations have demonstrated that antisense RNA is capable of reducing the expression of antibiotic genes that have been introduced into plant cells either transiently (Ecker and Davis, 1986) or stably (Rothstein et al., 1987; Delauney et al., 1988). An antisense gene for chalcone synthase has also been found to be effective in altering flower pigmentation (van der Krol et al., 1988) and transgenic plants expressing the potato virus X (PVX) coat protein appear to afford low levels of protection against infection by PVX (Hemenway et al., 1988). Our results demonstrate that antisense RNA is capable of sharply reducing the accumulation of one of the most plentiful leaf mRNA species (Tobin and Silverthorne, 1985) and the most abundant soluble protein in plant cells (Ellis, 1979). Therefore, antisense RNA technology seems to be a promising means for inhibiting the expression of any plant gene, regardless of its expression level. This technology may be of general utility in generating mutants to attack fundamental problems in basic and applied plant biology. Such an approach seems especially appropriate for overcoming the difficulties associated with classical selection procedures for mutations in genes that are members of multigene families.

;;isense

mRNA Inhibition

Experimental

of RUBISCO

Levels

Procedures

Agrobacterium-Mediated Transformations and Growth of Plant Material The rbcS antisense DNA construct (pTASS) was introduced by the triparental mating procedure into a strain of Agrobacterium tumefaciens harboring the disarmed Ti plasmid pGV2260 (Deblaere et al., 1965); GJ23 served as the E. coli mobilizing strain in these conjugations (Van Haute et al., 1963). Exconjugants expressing the appropriate antibiotic resistances were selected and used to infect leaf discs of Nicotiana tabacum cultivar Petit Havana SRl (Maliga et al., 1973) by procedures described in Horsch et al. (1985). The infected discs were then placed on tissue culture medium containing kanamycin (50 &ml) (Horsch et al., 1985). After several weeks, putative transformants were removed from the disks and propagated in tissue culture under nonselective conditions on 85 medium (Horsch et al., 1985) sup plemented with 1% sucrose (27oC, 16 hr day, 6 hr night, 520 uEinsteins M2 set’). Five kanamycin-resistant transgenic plants were randomly selected for further investigation: these have been designated transformants “3,” “5,” “7:’ “23:’ and “25.” As controls, untransformed SRI plants were germinated from seed on 85 tissue culture medium supplemented with 1% sucrose. Three of these SRl plants were randomly selected and propagated on sucrose-containing tissue culture medium under the same conditions as the transformed plants; these controls have been designated plants “A,” “C,” and “E.” Under these conditions, some of the transformed plants grew somewhat more slowly than the control plants, and some of the transformants were also more pale and had thinner stems and less broad leaves than the control plants. Some untransformed SRl seeds, as well as seeds from the self-fertilization of transformant 5, were germinated in vermiculite and maintained in a growth chamber (28OC, 14 hr day, 10 hr night, 500 uEinsteins Mz seer) and watered daily with l/4 strength Hoagland’s solution. Preparation of DNA, RNA, and Protein The top three (expanding) leaves of plants maintained on tissue culture medium supplemented with 1% sucrose served as the source of nucleic acids and soluble proteins. These plants were approximately the same age and height, and each of the three leaves was approximately the same size from plant to plant. Genomic DNA was isolated by procedures described by Dellaporta et al. (1983) and DNA amounts were determined by the diphenylamine assay (described in Munro and Fleck, 1966). RNA was isolated by methods described in Bogorad et al. (1983). To isolate soluble proteins, the leaves were homogenized in a microfuge tube on ice by a hand-held pestle in a solution containing 100 m M Tris-HCI (pH 7.5) 100 m M NaCI, 5 m M EDTA, 10 m M f3-mercaptoethanol, and 2.5 m M PMSF (phenlymethylsulfonyl fluoride). The samples were centrifuged for 10 min, and an aliquot of each supernatant was removed for Bradford protein assays (instructions provided by Pierce Chemical). SDS (sodium dodecyl sulfate) and DTT (dithiothreitol) were immediately added to the remainder of each supernatant to final concentrations of 2% and 50 mM, respectively, and the samoles were stored at -2oOC until use. Hybridization Analyses For Southern hybridizations (Southern, 1975) samples of DNA were digested with an appropriate restriction enzyme, electrophoresed through a 08% TBE agarose gel, and transferred to nylon filters (Gene Screen, NEN) by procedures described in Maniatis et al. (1982). The filters were hybridized and washed by methods described in Church and Gilbert (1984) using nick-translated (Maniatis et al., 1982) or oligolabeled (Pharmacia) probes at a concentration of about 5 x lo5 dpm/ml. For Northern hybridizations, samples of RNA were electrophoresed through a 1.2% MOPS-formaldehyde gel and transferred to nylon filters (Gene Screen, NEN) also by procedures described in Maniatis et al. (1962). The filters were then hybridized and washed using the same conditions as those described for Southern hybridizations. SDS-Polyacrylamlde Gel Electrophoresis Protein samples were boiled for 10 min, loaded onto a discontinuous SDS-polyacrylamide gel, and electrophoresed for 12-15 hr at a con-

stant current of 25 mA. The separating gel contained 15% (w/v) acrylamide, 0.4% (w/v) bisacrylamide, 0.375 M Tris-HCI (pH 6.8) 0.1% (w/v) ammonium persulfate, and 0.05% (v/v) TEMED (N,N,N’,N’,tetramethylethylenediamine). The stacking gel consisted of 3% (w/v) acrylamide, 0.08% (w/v) bisacrylamide, 0.375 M Tris-HCI (pH 6.6) 0.125% (w/v) ammonium persulfate, and 0.06% (v/v) TEMED. The upper and lower reservoir buffers contained 50 m M Tris, 0.2 M glycine, and 0.2% SDS. After electrophoresis, the gel was stained with Coomassie blue. In Vivo Labeling of Proteins The second and third leaves of intact plants growing on tissue culture medium supplemented with 1% sucrose were spotted (in 1 ul aliquots) with 150 uCi of 3rS-methionine (NEN) in 10 ul of 1% Tween-20. After a labeling period of 1 hr under continuous illumination, the leaves were detached from the plant and the soluble proteins were isolated by procedures described above. An aliquot of each protein sample was used to measure TCA precipitable label (Dietz and Bogorad, 1987) and equal cpm were then electrophoresed through an SDS-polyacrylamide gel as described above. After electrophoresis, the gel was fixed and fluorographed (Dietz and Bogorad, 1967). Acknowledgments The authors wish to thank Sunanda Babu, Laila Banu, and Gisele Drouin for expert technical assistance. They would also like to thank S. Sugiuraand Leon Hirth for providing pTB5 and pSEM1, respectively, Ken Richards and Alice Cheung for providing some of the intermediates used in the construction of pTASS, and Daniel Voytas, Jean Lukens, and Alan Blowers for critical reading of the manuscript. This work was supported in part by a research grant from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received July 14, 1988: revised August 31, 1986. References Abbott, M. S., and Bogorad, L. (1987). Light regulation of genes for the large and small subunits of ribulose-bisphosphate carboxylase in tobacco. In Progress in Photosynthesis Research, Vol. IV, J. Biggins, ed. (Dordrecht, The Netherlands: Martinus Nijhoff Publishers), pp. 527-530. Barraclough, R., and Ellis, R. J. (1980). Assembly of newly synthesized large subunits into ribulose bisphosphate carboxylase in isolated pea chloroplasts. Biochim. Biophys. Acta 608, 19-31. Bedbrook, J. R., Coen, D. M., Beaton, A., Bogorad, L., and Rich, A. (1979). Location of the single gene for the large subunit of ribulosebisphosphate carboxylase on the maize chloroplast chromosome. J. Biol. Chem. 254, 905-910. Berry, J. O., Nikolau, B. J., Carr, J. l?, and Klessig, D. F. (1965). panscriptional and post-transcriptional regulation of ribulose 1,5-bisphosphate carboxylase gene expression in light- and dark-grown amaranth cotyledons. Mol. Cell. Biol. 5, 2238-2246. Berry, J. O., Nikolau, B. J., Carr, J. l?, and Klessig, D. F. (1966). Translational regulation of light-induced ribulose l$bisphosphate carboxylase gene expression in amaranth. Mol. Cell. Biol. 6, 2347-2353. Bevan, M. W., Mason, S. E., and Goelet, P (1985). Expression of tobacco mosaic virus coat protein by a cauliflower mosaic virus promoter in plants transformed by Agrubactedum. EMBO J. 4, 1921-1926. Bogorad, L. (1982). Regulation of intracellular gene flow in the evolution of eukaryotic genomes. In On the Origins of Chloroplasts, J. A. Schiff, ed. (Amsterdam: Elsevier/North Holland), pp. 277-295. Bogorad, L., Gubbins, E. J., Krebbers, E., Larrinua, I. M., Mulligan, B. J., Muskavitch, K. M. T., Orr, E. A., Rodermel, S. R., Schantz, R., Steinmetz, A. A., De Vos, G., and Ye, Y. Y. (1983). Cloning and physical mapping of maize plastid genes. Meth. Enzymol. 97, 524-554.

Cannon, S., Wang, i?, and Roy, H. (1966). Inhibition of ribulose bisphosphate carboxylase assembly by antibody to a binding protein. J. Cell Biol. 103, 1327-1335.

Mazur, B. J., and Chui, C.-F. (1965). Sequence of a genomic DNA clone for the small subunit of ribulose bis-phosphate carboxylase-oxygenase from tobacco. Nucl. Acids Res. 73, 2373-2386.

Church, G.. and Gilbert, W. (1964). Genomic sequencing. Acad. Sci. USA 81, 19911995.

Miziorko, H. M., and Lorimer, G. H. (1963). Ribulose-1,5-bisphosphate carboxylase-oxygenase. Annu. Rev. Biochem. 52, 507-535.

Proc. Natl.

Coen, D. M., Bedbrook, J. R., Bogorad, L., and Rich, A. (1977). Maize chloroplast DNA fragment encoding the large subunit of ribulosebisphosphate carboxylase. Proc. Natl. Acad. Sci. USA 74, 5467-5491.

Munro, H. N., and Fleck, A. (1966). The determination of nucleic acids. In Methods of Biochemical Analysis, Vol. XIV, D. Glick, ed. (New York: John Wiley and Sons), pp. 113-176.

Deblaere, Ft., Bytebier, B., De Greve, H., Deboeck, F., Schell, J., Van Montagu, M., and Leemans, J. (1965). Efficient octopine Ti plasmidderived vectors for Agro&xscferium-mediated gene transfer to plants. Nucl. Acids Res. 13, 4777-4768.

Musgrove, J. E., Johnson, R. A., and Ellis, R. J. (1987). Dissociation of the ribulosebisphosphate-carboxylase large-subunit binding protein into dissimilar subunits. Eur. J. Biochem. 763, 529-534.

Delauney, A. J., Tabaeizadeh, Z., and Verma, D. P S. (1966). A stable bifunctional antisense transcript inhibiting gene expression in transgenie plants. Proc. Natl. Acad. Sci. USA 85, 4300-4304. Dellaporta, S. L., Wood, J., and Hicks, J. B. (1983). A plant DNA minipreparation: version II. Plant Mol. Biol. Reporter 1, 19-21. Dietz, K.-J., and Bogorad, L. (1967). Plastid development in Pisum setivum leaves during greening. I. A comparison of plastid polypeptide composition and in organello translation characteristics. Plant Physiol. 85, 606-815. Ecker. J. R., and Davis, R. W. (1986). Inhibition of gene expression in plant cells by expression of antisense RNA. Proc. Natl. Acad. Sci. USA 83, 5372-5376. Ellis, R. J. (1979). The most abundant protein in the world. Trends Biothem. Sci. 4, 241-244. Guilley, H., Dudley, R. K., Jonard, G., Balazs, E., and Richards, K. E. (1962). Transcription of cauliflower mosaic virus DNA: detection of promoter sequences, and characterization of transcripts. Cell 30, 763-773. Hain, R., Stabel, P, Czernilofsky, A. f?, Steinbiss, H. H., HerreraEstrella, L., and Schell, J. (1985). Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts. Mol. Gen. Genet. 199, 161168. Hattori, T., and Margulies, M. M. (1986). Synthesis of large subunit of ribulosebisphosphate carboxylase by thylakoid-bound polyribosomes from spinach chloroplasts. Arch. Biochem. Biophys. 244, 630-640. Hemenway, C.. Fang, R.-X., Kaniewski, W. K., Chua, N.-H., and Turner, N. E. (1966). Analysis of the mechanism of protection in transgenic plants expressing the potato virus coat protein or its antisense RNA. EMBO J. 7, 1273-1280. Hemmingsen, S. M., and Ellis, R. J. (1966). Purification and properties of ribulosebisphosphate carboxylase large subunit binding protein. Plant Physiol. 80, 269-276. Hildebrandt, J., Bottomley, W.. Moser, J., and Herrmann, R. G. (1964). A plastome mutant of Denothers hookeri has a lesion in the gene for the large subunit of ribulose-l,&bisphosphate carboxylase/oxygenase. Biochim. Biophys. Acta 783, 67-73. Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. G., and Fraley, R. T (1965). A simple and general method for transferring genes into plants. Science 227, 1229-1231. Inamine, G., Nash, B., Weissbach, H., and Brot, N. (1965). Light regulation of the synthesis of the large subunit of ribulose-ld-bisphosphate carboxylase in peas: evidence for translational control. Proc. Natl. Acad. Sci. USA 82, 5690-5694. Kirk, M. M., and Kirk, D. L. (1965). Translational regulation of protein synthesis, In response to light, at a critical stage of Volvox development. Cell 47, 419-428. Kuhlemeier, C., Green, P J., and Chua, N.-H. (1967). Regulation of gene expression in higher plants. Annu. Rev. Plant Physiol. 38, 221-257. Maliga, P, Sz.-Breznovits, A., and Marton, L. (1973). Streptomycinresistant plants from callus culture of haploid tobacco. Nature New Biol. 244, 29-30. Maniatis, T., Fritsch, E. F, Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory).

Nikolau, B. J., and Klessig, D. F. (1987). Coordinate, organ-specific and developmental regulation of ribulose 1,5-bisphosphate carboxylase gene expression in Amaran8x~s hypochondriacus. Plant Physiol. 85, 167-173. Odell, J. T., Nagy, F., and Chua, N.-H. (1985). Identification of DNA sequences required for activity of the cauliflower mosaic virus 35s promoter. Nature 313, 610-612. O’Neal, J. K., Pokalsky, A. R., Kiehne, K. L., and Shewmaker, C. K. (1967). Isolation of tobacco SSU genes: characterization of a transcriptionally active pseudogene. Nucl. Acids Res. 75, 8661-6677. Pinck, M., Guilley, E., Durr, A., Hoff, M., Pinck, L., and Fleck, J. (1984). Complete sequence of one of the mRNAs coding for the small subunit of ribulose bisphosphate carboxylase of Nicotiene sylvestris. Biochimie 88, 539-545. Radetzky, R., and Zetsche, K. (1987). Effects of specific inhibitors on the coordination of the concentrations of ribulose-bisphosphate-carboxylase subunits and their corresponding mRNAs in the alga Cblorogonium. Planta 172, 36-46. Rothstein, S. J., DiMaio, J., Strand, M., and Rice, D. (1967). Stable and heritable inhibition of the expression of nopaline synthase in tobacco expressing antisense RNA. Proc. Natl. Acad. Sci. USA 84,843~6443. Roy, H., Bloom, M., Miles. l?, and Monroe, M. (1962). Studies on the assembly of large subunits of ribulose bisphosphate carboxylase in isolated pea chloroplasts. J. Cell Biol. 94, 20-27. Sanders, P R., Winter, J. A., Barnason, A. R., Rogers, S. G., and Fraley, R. T. (1967). Comparison of cauliflower mosaic virus 35s and nopaline synthase promoters in transgenic plants. Nucl. Acids Res. 75, 1543-1556. Sasaki, Y. (1966). Effects of alpha-amanitin on coordination of two mRNAs of ribulose-bisphosphate carboxylase in greening pea leaves. FEBS Lett. 204, 279-262. Sasaki, Y., Nakamura, Y., and Matsuno, R. (1967). Regulation of gene expression of ribulose bisphosphate carboxylase in greening pea leaves. Plant Mol. Biol. 8, 375-382. Schmidt, G. W., and Mishkind, M. L. (1983). Rapid degradation of unassembled ribulose Id-bisphosphate carboxylase small subunits in chloroplasts. Proc. Natl. Acad. Sci. USA 80, 2632-2636. Schmidt, G. W., and Mishkind, M. L. (1986). The transport of proteins into chloroplasts. Annu. Rev. Biochem. 55, 879-912. Sheen, J.-Y, and Bogorad, L. (1966). Expression of the ribulose-1,s bisphosphate carboxylase large subunit gene and three small subunit genes in two cell types of maize leaves. EMBO J. 5, 3417-3422. Shinozaki, K., and Sugiura, M. (1962). The nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose-ld-bisphosphate carboxylaseloxygenase. Gene 20, 91-102. Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. Spreitzer, R. J., Goldschmidt-Clermont. M., Rahire, M., and Rochaix, J.-D. (1965). Nonsense mutations in the Cblemydomones chloroplast gene that codes for the large subunit of ribulosebisphosphate carboxylase/oxygenase. Proc. Natl. Acad. Sci. USA 82, 5460-5464. Steinbiss, H. J., and Zetsche, K. (1966). Light and metabolic regulation of the synthesis of ribulose-1.5bisphosphate carboxylaseloxygenase and the corresponding mRNAs in the unicellular alga Chlomgonium. Planta 187, 575-581.

Antisense 681

mRNA Inhibition

of RUBISCO

Levels

Tobin, E. M., and Silverthorne, J. (1985). Light regulation of gene expression in higher plants. Annu. Rev. Plant Physiol. 36, 569-593. Towbin, H., Staehelin, T, and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4359-4354. van der Krol, A. R., Lenting, P E., Veenstra, J., van der Meer, I. M., Koes, R. E., Gerats, A. G. M., Mel, J. N. M., and Stuitje, A. R. (1988). An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentatation. Nature 333, 866-869. Van Haute, E., Joos, H., Maes, M., Warren, G., Van Montagu, M., and Schell, J. (1983). Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J. 2, 411-417.