AU2006249249B2 - Plastid promoters for transgene expression in the plastids of higher plants - Google Patents

Description

PLASTID PROMOTERS FOR TRANSGENE EXPRESSION IN THE PLASTIDS OF HIGHER PLANTS Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from the National Science Foundation, Grant Number MCB96-30763. FIELD OF THE INVENTION The present invention relates to plant genetic engineering and particularly to plastid transformation in 5 higher plants. The invention provides novel promoter sequences useful for the expression of foreign genes of interest in various plant species. BACKGROUND OF THE INVENTION 10 Chloroplast genes are transcribed by an RNA polymerase containing plastid-encoded subunits homologous to the a, # and 3' subunits of E. coli RNA polymerase. The promoters utilized by this enzyme are similar to E. coli a 70 - promoters consisting of -35 and -10 consensus 15 elements (G.L. Igloi and H. Kossel, Crit. Rev. Plant Sci. 10, 525, 1992; W. Gruissem and J.C. Tonkyn, Crit. Rev. Plant. Sci. 12:-19, 1993) Promoter selection by the plastid-encoded RNA polymerase is dependent on nuclear encoded sigma-like factors (Link et al. 1994, Plant 20 promoters and transcription factors, Springer Verlag, Heidelberg, pp 63-83). In addition, transcription activity from some promoters is modulated by nuclear encoded transcription factors interacting with elements upstream of the core promoter (L.A. Allison and P. 25 Maliga, EMBO J., 14:3721-3730; R. Iratni, L. Baeza, A. Andreeva, R. Mache, S. Lerbs-Mache, Genes Dev. 8, 2928, 1994, Sun et al., Mol. Cell Biol. 9:5650-5659, 1989). These factors mediate nuclear control of plastid gene expression in response to developmental and environmental -2 5 stimuli. The existence of a second nuclear encoded polymerase transcription system in plastids has been demonstrated. However, the relevant nucleic acid sequences required for transcription initiation 10 comprising the novel regulatory elements of this system have yet to be elucidated. It is an object of the present invention to provide these novel genetic elements. Incorporation of these regulatory elements into specific plastid directed DNA constructs enables 15 greater flexibility and range in plant species available for plastid transformation, and facilitates ubiquitous expression of foreign proteins and/or RNAs and are useful in non-green plastids. 20 SUMMARY OF THE INVENTION Promoters contain distinct DNA sequence information to facilitate recognition by the RNA polymerase and initiation of transcription leading to gene expression. In accordance with the present 25 invention, promoters have been discovered which function in both monocots and dicots. These promoter elements may be used to advantage to express foreign genes of interest in a wider range of plant species. Additionally, the promoter elements of the invention drive expression of 30 exogenous genes in non-green tissues. It is an object of the present invention to provide DNA constructs and methods for stably transforming plastids of multicellular plants containing such promoters. The DNA constructs of the invention extend the range of plant species that may 35 be transformed. The promoters recognized by plastid-encoded plastid RNA (PEP) polymerase have been well characterized in photosynthetic tissues such as leaf. The utility of PEP promoters for expression of foreign proteins in non 40 green tissues is demonstrated herein. The nuclear-encoded plastid (NEP) polymerase transcription system of the - 3 5 present invention directs expression of plastid genes also in roots, seeds, meristematic tissue and/or leaves. In most plants, including maize, cotton and wheat, plant regeneration is accomplished through somatic embryogenesis (i.e., involving meristematic tissue). In 10 a preferred embodiment of the invention, efficient plastid transformation in these crops will be greatly facilitated, through the use of the NEP and PEP plastid transcription system and promoters of the present invention. 15 Particularly preferred promoters for use in the constructs of the invention are the clpP -111 (SEQ ID NOS: 15, 16, 30 and 31) promoters for the transformation of monocots and dicots and the pclp -53 promoter for transformation in dicots. Homologous clpP promoters from 20 other plant species are contemplated to be within the scope of the present invention. Other preferred promoters for use in expressing foreign genes of interest in the plant plastid in non green tissues are PEP promoters present in the 16SrDNA 25 operon, SEQ ID NOS: 28 and 29. Additional promoter elements suitable for use in the present invention are the rpoB and atpB promoters. The NEP promoters of the invention are incorporated into currently available plastid 30 transformation vectors such as those described in pending U.S. Application No. 08/189,256, and also described by Svab & Maliga., Proc. Natl. Acad. Sci. USA, 90, 913 (1993). Protocols for using such vectors are described in U.S. Patent No. 5,451,513. The disclosures of the 35 three references cited above are all incorporated by reference herein. To obtain transgenic plants, plastids of non-photosynthetic tissues are transformed with selectable marker genes expressed from NEP promoters and transcribed by the nuclear-encoded polymerase. Likewise, 40 to express foreign proteins of interest, expression cassettes are constructed for high level expression in -4 5 non-photosynthetic tissue, using the NEP promoter transcribed by the nuclear-encoded plastid RNA polymerase. In another aspect of the invention, PEP promoters of the invention are incorporated into currently available plastid transformation vectors and 10 protocols for use thereof. In yet another aspect of the invention, the NEP transcription system also may be combined with the ac 7 type system through the use of dual NEP/PEP promoters. In transforming DNA constructs, the promoters are arrayed 15 in tandem, operably linked to the coding region of the foreign gene of interest. As used herein, the term transcription unit refers to isolated DNA segments which comprise the essential coding regions of one or more exogenous protein(s) of interest. Such transcription 20 units may also contain other cis elements for enhancing gene expression, such as enhancer elements. Transcription units are operably linked to the promoters of the invention, such that expression of the transcription unit is regulated by said promoter. 25 Particularly preferred promoters for use in combination are the Prrn PEP promoters combined with the clpP type II NEP promoter in dicots and the Prrn PEP promoter combined with the clpP Type I NEP promoter for use in both monocots and dicots. A suitable Prrn promoter has the 30 following sequence (SEQ ID NO: 32) 5'-GCTCCCCCGC CGTCGTTCAA TGAGAATGGA TAAGAGGCTC GTGGGATTGA CGTGAGGGGG CAGGGATGGC TATATTCTG GGAGCGAACT CCGGGCGAAT ACGAAGCGCt TGGATACAGT TGTAGGGAGG GATT-3'. Homologous PEP and NEP promoters from a variety 35 of plant species corresponding to those listed above are also considered to fall within the scope of the present invention. The transforming DNA also contains 3' regulatory regions of plant or bacterial origin to effect efficient termination of transcription. An exemplary 3' 40 regulatory region is shown in Figure 13, SEQ ID NO: 27.

-5 5 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts a series of autoradiographs illustrating RNA steady state concentrations in green (G) and white (W) iojap maize leaves. To control for loading, blots shown above were reprobed for cytoplasmic 10 25S ribosomal RNA as described (Dempsey et al., Mol. Plant Path. 83:1021-1029, 1993) (lower panels). Figures 2A and 2B depict a pair of autoradiographs showing the results of primer extension 15 analysis on green (G) and.white (W) iojap maize leaves for the mapping of the clpP promoter, Fig. 2A. The number -111 refers to the transcript 5' end relative to the ATG translation initiation codon. Figure 2B is an autoradiograph showing the results of an in vitro capping 20 and RNase protection assay to identify primary transcript 5' ends. Note that the RNase protection construct is short and protects only a 79 nt fragment. Size of molecular weight (MW) markers (100, 200, 300, 400, and 500 nucleotides) is also shown. 25 Figures 3A and 3B show the mapping of maize rpoB promoters. Fig. 3A is an autoradiograph showing the results of primer extension analysis of RNA from green (G) and white (W) iojap maize leaves. The number -147 30 refers to transcript 5'-end. Fig. 3B shows the results of an in vitro capping and RNase protection assay to identify primary transcript 5'-ends. Note that the RNase protection construct is short and protects only a 74 nt fragment. Size of molecular weight (MW) markers (100, 35 200, 300, 400, and 500 nucleotides) is also shown. Figures 4A, 4B and 4C show the mapping of atpB promoters. Fig. 4A show the results of primer extension analysis of RNA from green (G) and white (W) iojap maize 40 leaves. Number -298 and -601 refer to transcript 5'-ends. Fig. 4B shows the results of an in vitro capping and - 6 5 RNase protection assay to identify primary transcript 5' ends. Note that the RNase protection construct is short and protects only a 79 nt fragment. Size of molecular weight (MW) markers (100, 200, 300, 400, and 500 nucleotides) is also shown. Fig. 4C depicts a physical 10 map of the atpB - rbcL intergenic region. Map position of the primary transcript 5' ends for NEP and PEP promoters are marked with filled and open circles, respectively. Figure 5A shows the alignment of DNA sequences 15 flanking the NEP promoter transcription initiation sites. Site of transcription initiation is marked (filled circles). Regions with significant similarity are boxed (Box I and Box II). Sequences corresponding to the loose 10-nt dicot NEP consensus are underlined with thin lines. 20 clpP -111 promoter region is maize is indicated by the thick underlining. Figure 5A shows rpoB (SEQ ID NO: 1), atpB (SEQ ID NO: 2) and clpP (SEQ ID NO: 3) promoter regions, respectively in maize. The alignment and nucleotide sequences of atpB promoters in maize, sorghum, 25 barley, wheat, and rice are shown in Figure 5B and correspond to SEQ ID NOS: 4, 5, 6, 7 and 8 respectively. Figure 5C shows the alignment of the rpoB sequences in maize (SEQ ID NO: 9), rice (SEQ ID NO: 10) and tobacco (SEQ ID NO:ll). The alignment of the clpP sequences in 30 maize (SEQ ID NO: 12), rice (SEQ ID NO: 13), and wheat (SEQ ID NO: 14 is shown in Figure 5D. Figure 6 shows the sequence alignment of the tobacco (SEQ ID NO: 15) and rice (SEQ ID NO: 16) plastid 35 clpP promoter regions. The NEP transcription initiation sites are marked with filled circles, the PEP transcription initiation site is marked with an open circle. The third tobacco NEP promoter is outside the sequence shown. The clpP coding regions are boxed. The 40 29-bp shared homologous region around the Type II tobacco PclpP-53 promoter is underlined.

-7 5 Figure 7 is a schematic drawing of the plastid targeting region of plasmid pDS44. On top is shown the restriction map of the chimeric uidA gene. PclpP is the rice clpP promoter fragment between the SacI and Nco site (SEQ ID NO: 17) which is shown at the bottom of Fig. 5. 10 uidA encodes the 0-glucuronidase reporter enzyme. Trpsl6 is the rps16 ribosomal protein gene 3' untranslated region. A map of the transforming DNA in a pPRV111A plastid vector (Gene Bank Accession No. U12812) is also shown (Zoubenko et al., Nucleic Acids Res. 22:3819-3824, 15 1994). Figures BA and 8B illustrate the promoter activity of the rice clpP promoter region in transgenic tobacco plastids. Figure 8A shows the results of primer 20 extension analysis to map RNA 5'-ends upstream of the rice PclpP::uidA::Trpsl6 chimeric gene. Primer extension analysis was carried out on total cellular RNAs isolated from the leaves of wild-type (wt) and transplastomic (T) tobacco plants. The numbers (-61, -111, -136, -169, 177) 25 refer to nucleotide positions of the mapped 5'ends relative to the ATG translation initiation codon of the rice clpP gene. Figure 8B shows a schematic representation of RNA 5' ends mapped in the rice clpP promoter region in rice (Os) and in tobacco (Os in Nt), 30 and upstream of the wild-type tobacco clpP gene (Nt). Promoters identified in homologous systems are marked (NEP, filled circles, PEP, open circles). Numbers indicate distance from translation initiation codon (nucleotide upstream of ATG is position -1). 35 Figure 9 depicts an alignment of DNA sequences which are conserved around the plastid clpP transcription initiation sites. Sequences are aligned for Marchantia polymorpha (Kochi) (SEQ ID NO: 18), Pinus contorta 40 (Clarke)(SEQ ID NO: 19), spinach (Westhof) (SEQ ID NO: 20), tobacco (Hajdukiewicz et al., 1977) (SEQ ID NO: 21), - 8 5 rice (SEQ ID NO: 22), maize (SEQ ID NO: 23) and Arabidopsis (SEQ ID NO: 24). Wild-type RNA 5' ends are marked with filled circles. The -61 5' end of the rice clpP-uidA chimeric mRNA is marked with an asterisk. Conserved nucleotides are boxed if identical at least in 10 five species. The translation start codons are shaded. Figure 10 shows the plastid targeting region of plasmid pPS18. Plasmid pPS18 has the plastid targeting region of plastid vector pPRV111A, GenBank Accession No: 15 U12812, with a chimeric uidA gene expressed from the PclpP-53(-22/+2 5 ) promoter. DNA sequence of the uidA gene is shown in Figure 13 (SEQ ID NO: 27). Figure 11 depicts the clpP 5'fragments tested 20 for promoter activity. The largest segment contains the PclpP-53 and PclpP-95 transcription initiation sites derived from Type II NEP and PEP promoters, respectively. The boundaries give the distance in nucleotides from PclpP-53 transcription initiation site (+1). SEQ ID NO: 25 25 contains the sequences present between -22 and +25 and SEQ ID NO: 26 shows the nucleotides present between -10 and +25. Both sequences function as promoters. Figure 12 depicts the results of primer 30 extension analysis to test promoter activity of clpP 5' fragments included in Figure 11. Transcripts derived from PclpP-53 (-53), a minor NEP promoter (*), PclpP-95 (-95) and PclpP-173 (-173) are marked. DNA sequences on the side give distance from primer in nucleotides. 35 Figure 13 shows the DNA sequence (SEQ ID NO: 27) of the chimeric uidA gene. SacI and HindIII cloning sites are marked. SacI, XhoI, NcoI, XbaI and HindIII sites are underlined. Translation initiation (ATG) and 40 stop (TGA) codons are underlined twice. Nucleotides derived from the tobacco plastid genome are in capital -9 5 letters; the position of the first and last nucleotide within the genome is listed. Figure 14 is a northern blot showing RNA steady state concentrations in leaf (L) and in embryogenic 10 cultured cells (E) of rice. To control for loading, the blots were stripped and probed for cytoplasmic 25S ribosomal RNA (lower panels). Hybridization signals were quantified with a Molecular Dynamics PhosphorImager. The fold excesses in leaves over cultured cell signals are 15 shown below the panels. Figure 15 is a Southern blot showing the relative plastid genome copy number in leaves (L) and in cultured embryogenic cells (E) of rice. EcoRI-digested 20 total cellular DNA (approximately 2 pg per lane) was probed for plastid 16SrDNA (upper) and nuclear-encoded 25SrDNA (lower). Hybridization signals were quantified with a Molecular Dynamics PhosphorImager. The ratio of 16SrDNA/25SrDNA signal intensity in leaves relative to 25 embryogenic cells was 1.1. Figure 16 is an autoradiograph depicting the mapping results of plastid mRNA promoters in rice leaves (L) and embryogenic cultured cells (E) using primer 30 extension analysis. Numbers on the right indicate the distance between the translation initiation codon (ATG) and 5' ends of primary transcripts (PEP, 0; NEP,@), or of processed mRNAs (-). DNA sequences on -the left are size markers. Sequence ladders shown for 16S rDNA and clpP 35 were obtained with homologous template and oligonucleotides used for primer extension analysis. Figure 17 shows the alignment of the maize (SEQ ID NO: 28 and rice (SEQ ID NO: 29) PEP promoter in the 40 rrn operon, Figure 17A. Figure 17B shows the alignment of the plastid clpP promoter regions in maize (SEQ ID NO: - 10 5 30) with the homologous regions in rice (SEQ ID NO: 31). PEP (0) and NEP (0) transcription initiation sites and processed 5' ends (-) are marked. For 16SrDNA sequence information see Strittmatter et al. (1985). The present invention provides sequence information for maize clpP. LO DESCRIPTION OF THE INVENTION Several reports have suggested the existence of an additional plastid-localized, nuclear-encoded RNA polymerase (reviewed in Gruissem and Tonkyn, 1993; Igloi L5 and Kossel, 1992; Mullet, 1993; Link, 1994). By deleting the rpoB gene encoding the essential 3 subunit of the tobacco E. coli-like RNA polymerase, the existence of a second plastid transcription system which is encoded by the nucleus has been established (Allison 20 et al., 1996, EMBO J. 15:2802-2809). Deletion of rpoB yielded photosynthetically defective, pigment-deficient plants. While the activity of the previously-known plastid-encoded a 70 -type transcription system in 25 photosynthetically active tissues, such as leaf, has been the subject of much research, the nuclear-encoded polymerase transcription system has not yet been characterized. In accordance with the present invention, it has been discovered that the NEP system also directs 30 expression in roots, seeds and meristematic tissue. In most plants, including maize, cotton and wheat, plant regeneration is accomplished through somatic embryogenesis (i.e., involving meristematic tissue). Efficient plastid transformation in these crops will be 35 enabled, or greatly facilitated, through the use of clpP promoters driven by the NEP plastid transcription system of the present invention. The ATP dependent Clp protease is widespread, if not ubiquitous, among both procaryotic and eucaryotic 40 cells (Goldberg, Eur. J. Biochem. 203:9-23, 1992). Clp protease expression has recently been examined in - 11 5 chlamydomonas (Huang et al. Mol. Gen. Genetics 244:151 159, 1994). The results of gene knockout experiments in this unicellular algae demonstrated the following: 1) all the transformants were found to be heteroplasmic mutants containing both the disrupted clpP and wild-type 10 copies; 2) the transformants persisted as heteroplasmic mutants after six rounds of growth and screening under selection pressure for the disrupted clpP; and 3) the heteroplasmic mutant stabilized at a level where approximately 80% of the clpP DNA copies were disrupted. 15 These data indicate that the clpP is essential for cell growth even under conditions where photosynthesis is not required. Shanklin et al. have examined the immunolocalization of clpP protein in Arabidopsis 20 chloroplasts (Shanklin et al. The Plant Cell 7:1713-1722, 1995). These studies revealed that clpP and clpC are constitutively expressed in all tissues of Arabidopsis at levels equivalent to those of E. coli clpP and clpA. The observation that the clpP NEP promoter drives 25 constitutive gene expression in all parts of a plant makes this promoter particularly suitable for use in the present invention. The NEP promoters of the invention are incorporated into currently available plastid 30 transformation vectors such as those described in pending U.S. Application No. 08/189,256, and also described by Svab & Maliga., Proc. Natl. Acad. Sci. USA, 90, 913 (1993). Protocols for using such vectors are described in U.S. Patent No. 5,451,513. The disclosures of the 35 three references cited above are all incorporated by reference herein. To obtain transgenic plants, plastids of non-photosynthetic tissues as well as photosynthetic tissues are transformed with selectable marker genes expressed from NEP promoters and transcribed by the 40 nuclear-encoded polymerase. Likewise, to produce foreign proteins of interest, expression cassettes are - 12 5 constructed for high level expression in non photosynthetic tissue, using the clpP NEP promoter transcribed by the nuclear-encoded plastid RNA polymerase. The NEP transcription system also may be combined with the a 70 -type system through the use of dual 10 NEP/PEP promoters. For versatility and universal applications, expression of selectable marker genes for plastid transformation is desirable in all targeted tissue types at a high level. Selectable marker genes in the 15 currently utilized plastid transformation vectors are expressed from PEP promoters recognized by the plastid encoded RNA polymerase. The PEP polymerase transcribes photosynthetic genes and some of the housekeeping genes, and therefore appears to be the dominant RNA polymerase 20 in photosynthetically active leaf tissues. Efficient plastid transformation has been achieved in tobacco based on chloroplast transformation in leaf cells. However, plant regeneration is not feasible, or is not practical from the leaves of most agronomically important cereal 25 crops, including maize, rice, wheat and in cotton. In these crops, transgenic plants are typically obtained by transforming embryogenic tissue culture cells or seedling tissue. Given that these tissues are non-photosynthetic, expression of marker genes by constitutive clpP NEP 30 promoters which are active in non-green tissues will facilitate transformation of plastids in all non-photosynthetic tissue types. Furthermore, as demonstrated herein, the ribosomal RNA operon PEP promoter is highly active in rice embryogenic cells. 35 The following nonlimiting Examples describe the invention in greater detail. Specifically, Examples I III below describe preferred methods for making and using the DNA constructs of the present invention and for practicing the methods of the invention. Any molecular 40 cloning and recombinant DNA techniques not specifically described are carried out by standard methods, as - 13 5 generally set forth, for example, in Ausubel (ed.), Current Protocols in Molecular Biology. John Wiley & Sons, Inc.(1994). LO EXAMPLE I Identification of promoters for the nuclear-encoded plastid RNA polymerase in the ribosome-deficient maize mutant iolap 15 As described previously, plastid promoters with conserved -10/-35 elements are well characterized for the plastid-encoded plastid RNA polymerase, PEP. Additionally, a ten-nucleotide promoter consensus was 20 reported for a second, nuclear encoded, plastid RNA polymerase, NEP, in tobacco, a dicot (Hajdukeiwicz et al. 1997, in press). In this Example, NEP promoters active in monocots are described. NEP promoter mapping was carried out in the plastid ribosome-less maize mutant 25 iojap which lacks PEP. These studies have revealed that atpB, an ATPase subunit gene, contains promoters for both NEP and PEP. In contrast, clpP, a protease subunit gene, and the rpoB operon, encoding the rpoB, rpoC1 and rpoC2 PEP subunit genes, are.transcribed from NEP promoters 30 only. These findings suggest conservation of transcription systems between monocots and dicots for the expression of certain plastid genes. The monocot NEP promoters share sequence homology around the transcription initiation site, including the 35 10-nucleotide loose consensus identified in dicots. In the plastids of photosynthetic higher plants, genes are transcribed by at least two RNA polymerases: the plastid-encoded plastid RNA polymerase (PEP) and the nuclear-encoded plastid RNA polymerase 40 (NEP). The sigma-factor homologues and PEP regulatory factors are encoded in the nucleus and are imported into plastids (Igoli and Kossel, (1992) supra; Link, (1996) Bioessays 18:465-471; Stern DB, Higgs DC, Yang J (1997) - 14 5 Trends Plant Sci 2:308-315). Much less is known about NEP. One appealing candidate for NEP is a 110 kD protein which has biochemical properties similar to yeast mitochondrial and T7 RNA polymerases. NEP may require accessory factors, such as CDF2 for its activity LO (Lerbs-Mache, 1993; Iratni et al., 94; Genes and dev). Dicot promoters for NEP were identified in tobacco plants lacking PEP due to deletion of rpoB encoding the PEP 1 subunit. The general rule emerging from these studies is that plastid genes fall into three L5 classes. Class I genes contain only PEP promoters. Examples of this class are the photosytem I and II genes. Class II genes contain both NEP and PEP promoters. Representative members of this class of genes are genes involved in plant metabolism and housekeeping genes. 20 Genes in the third class contain NEP promoters only. Genes in this class include rpoB and accD. As plastid transformation is not yet available in monocots, ArpoB plants could not be obtained for maize NEP promoter analysis. However, mutants with a defect in 25 plastid ribosome accumulation are available in barely (albostrians; Hess et al., EMBO J. 12:563-571, 1993) and maize (iojap; Walbot and Coe, Proc. Natl. Acad. Sci. 76:2760-2764, 1979; Han et al., Planta 191:552-563, 1993; Han et al., EMBO J. 11:4037-4046, 1992). In the absence 30 of plastid ribosomes, these mutants cannot synthesize PEP. Both mutants accumulate mRNAs for a subset of plastid genes indicating the presence of NEP activity (Hess et al., 1993, supra; Han et al., 1993, supra). NEP promoters for three plastid genes were 35 mapped in white ribosome-less maize iojap seedlings. The data show that the maize atpB gene has alternative NEP and PEP promoters while the clpP and the rpoB genes are transcribed from NEP promoters exclusively in both white and green seedlings. DNA sequence alignment 40 revealed that monocot NEP promoters share homology directly upstream of the transcription initiation site.

- 15 5 The homologous region includes the previously identified tobacco NEP promoter consensus elements suggesting conservation of the NEP transcription machinery between monocots and dicots. 10 Materials and Methods for Example I Plant Materials. Iojap is a recessive striped mutant of maize. Maternal white and green seedlings were obtained by crossing a striped ij/ij maternal parent (1404) with pollen from a wild type male (inbreed Oh5la). 15 The seeds were kindly provided by Rob Martienssen and Mary Byrne, Cold Spring Harbor Laboratory. Surface-sterilized seeds were germinated in vitro on 2% MS medium (24 *C, 16 hours illumination). 20 RNA Gel blots. Total cellular RNA was prepared from the leaves of 9-day-old seedlings (Stikema et al. Plant Mol. Biol. 11:255-269, 1988). The RNA (5 pg per lane) was electrophoresed on 1% agarose/formaldehyde gels, then transferred to Hybond N (Amersham) using the 25 Posiblot Transfer apparatus (Stratagene). Hybridization to random-primer labeled fragment was carried out in Rapid Hybridization Buffer (Amersham) overnight at 65 OC. Double-stranded ptDNA probes were prepared by random-primed 32 P-labeling of PCR-generated or 30 gel-purified DNA fragments. The sequence of the primers used for PCR, along with their positions within the tobacco (N.t.; Genebank Accession No. Z00044) or maize (Z.m.; Genebank Accession No. X86563) ptDNA are as follows: 35 Gene 5' nt position Sequence SEQ ID NO: in ptDNA atpB (Z.m.) 55860(C) GAGAGGAATGGAAGTGATTGACA (33) 55103 GAGCAGGGTCGGTCAAATC (34) 40 clpP (Z.m.) 69840 ATCCTAGCGTGAGGGAATGCTA (35) 70064(C) AGGTCTGATGGTATATCTCAGTAT (36) psbA (N.t.) 1550(C) CGCTTCTGTAACTGG (37) 667 TGACTGTCAACTACAG (38) - 16 5 The following ptDNA fragments were used as probes: rbcL(N.t.), a BamHI fragment (nucleotides 58047 to 59285 in ptDNA); 16SrDNA (N.t.), EcoRI to EcoRV fragment (nucleotides 138447 to 140855 in the ptDNA); rpoB, HindIII fragment (nt 24291-24816). 10 Primer extension analysis. Primer extension reactions were carried out on 10 yg of total leaf RNA as described (Allison et al. EMBO J. 15:2802-2809, 1996). The primers are listed below, with nucleotide positions 15 in the published maize plastid genome sequence (Maier et al., J. Mol. Biol. 251:614-628, 1995). Underlined oligonucleotides (added to create cloning site) were also used to generate the capping constructs, in which case the position of the first nucleotide in the genome 20 sequence is positioned immediately following the underline. Gene 5' nt position Sequence SEQ ID NO: in ptDNA 25 clpP#1 70182 GGTACTTTTGGAACACCAATGGGCAT (39) atpB#1 56095 GAAGTAGTAGGATTGGTTCTCATAAT (40) atpB#2 56640 GGTCTAGAATTCCTATCGAATTCCTTC (41) rpoB#1 21545(C) GAATCTACAAAATCCCTCGAATTG (42) 30 rpoB#2 21418(C) ACTCTTCATCAATCCcTACG (43) (Note: C at 3'-end of the atpB#2 oligonucleotide is at nt position 56644; the published sequence has a 15 nucleotide deletion relative to the sequence we found). 35 Identification of primary transcripts by in vitro capping. Total leaf RNA (20 Mg for clpP or 100 pg for rpoB and atpB transcripts) from white seedlings was capped in the presence of 0.25 or 1.0 mCi (a- 32 P) GTP 40 (Kennell and Pring, Mol. Gen. Genet. 216:16-24, 1989). Labeled RNAs were detected by ribonuclease protection (Vera et al., Plant Mol. Biol. 19:309-311, 1992) using the RPAII kit (Ambion). To prepare the protecting - 17 5 complementary RNA, an appropriate segment of the plastid genome was PCR-amplified using the primers listed below. Gene 5' nt position Sequence SEQ ID NO: in ptDNA 10 clpP#2 70241 GGTCTAGACTACACTTTAATATGGA (44) clpP#3 70549(C) GGGAATTCTGTTTGTAAGAAGA (45) atpB#2 56640 GGTCTAGAATTCCTATCGAATTCCTTC (46) atpB#3 56832(C) GGCTCGAGGGACAACTCGATAGGATTAGG (47) 15 rpoB#3 21394(C) GGTCTAGAATCTAGCAATCATGGAATC (48) rpoB#4 21066 GGCTCGAGCGTGCTATTCTAAATCGT (49) The 5' primers set forth above were designed to add a Xba I restriction site (underlined) upstream of the 20 amplified fragment. The 3' primers were designed to add a XhoI (atpB , rpoB) or EcoRI (clpP) site (underlined) downstream of the amplified sequence. The amplified product was cloned after digestion with the appropriate restriction enzyme into a pBSKS+ vector (Stratagene). To 25 generate unlabeled RNA complementary to the 5' end of RNAs, the resulting plasmid was linearized with XhoI (atpB, rpoB) or EcoRI (clpP) and transcribed in a Megascript (Ambion) reaction with T7 RNA polymerase. Markers (100, 200, 300, 400, and 500 nucleotides) were 30 prepared with the RNA Century Markers Template Set (Ambion), following the manufacturer's protocol. DNA sequence analysis. DNA sequence analysis was carried out utilizing the Wisconsin Sequence Analysis 35 Package (Genetics Computer Group, Inc.). Results and Discussion Plastid transcript accumulation in the maternal 40 white and green maize plants. Lack of 16S rRNA accumulation in the white maize plants confirmed the reported lack of plastid ribosomes in the white iojap seedlings (Walbot and Coe - 18 5 1979, supra). The absence of rbcL and psbA mRNAs, known to be transcribed from PEP promoters, indicates that the maternal white plants indeed lack PEP activity. (Han et al., 1993, supra). See Figure 1. Transcript accumulation for three additional LO plastid genes: clpP, rpoB and atpB has been analyzed in iojap plants. High steady-state mRNA levels, indicating the presence of NEP promoters was reported for these genes in iojap maize (Han et al., 1993, supra), albostrians barley (Hess et al., 1993, supra) and ArpoB L5 tobacco (Hajdukiewicz et al., EMBO J., in press, 1997), respectively. Readily-detectable accumulation of mRNA in the ribosome-less iojap plants confirmed that active NEP promoters regulate expression for each of these genes (Fig. 1). 20 The clpP NEP promoter is efficiently transcribed in white and green seedlings. To identify the NEP promoters, transcript 5'-ends were mapped by primer extension analysis. To 25 distinguish between 5'-ends that represent transcripts from NEP promoters from those generated by RNA processing, the 5'-ends were capped using guanylyltransferase. For clpP, significant mRNA accumulation was 30 found in both white and green seedlings. Primer extension analysis identified only one major clpP 5'-end at nucleotide position -111 (the nucleotide upstream of the ATG being at -1 position). The clpP -111 transcript could be capped in vitro. See Figure 2B. These data 35 confirm that this 5' end is a primary transcript and also identifies the maize NEP promoter PclpP-111. The same 5'-end is observed in both white and green maize seedlings indicating that the same clpP promoter is active in chloroplasts as well as in the 40 nonphotosynthetic iojap plastids (Fig. 2A). Therefore, PclpP -111 is considered to be a constitutive promoter.

- 19 5 In rice, the clpP transcript 5'-end mapped to the same nucleotide indicating conservation of PclpP -111 in monocots (data not shown). The rpoB NEP promoter activity is enhanced in 10 iojap plastids. RNA gel blot analysis shows that rpoB mRNA accumulates to a detectable level only in white seedlings (Fig. 1). However, more sensitive primer extension analysis revealed that the same 5'-ends were present in 15 both white and green leaves. Two 5'-ends could be identified, a major band at nucleotide position -147, and a minor band at position -285 (Fig. 3A). The in vitro capping assay confirmed that the -147 RNA species is a primary transcript (Fig. 3B), and therefore the product 20 of PrpoB-147 promoter. The atpB gene is transcribed from a NEP promoter in white plants and from a PEP promoter in green seedlings. 25 There is substantial atpB mRNA accumulation in green leaves, while much less is found in the white iojap leaves (Fig. 1). Primer extension analysis of mRNA from green leaves identified a transcript with a 5'-end at nucleotide position -298 (Fig. 4A) confirming an earlier 30 report (Mullet et al., Plant Mol. Biol. 4:39-54, 1985). This -298 mRNA species was absent in leaf RNA isolated from white plants, indicating that the -298 mRNA is a PEP transcript. Instead, another atpB transcript was mapped to nucleotide position -601 (Fig. 4A). The difference in 35 the size of the two mRNAs is apparent on the RNA gel blot shown in Fig. 1. The -601 transcript could be capped in vitro by guanylyltransferase, indicating that it is a primary transcript (Fig. 4B). Therefore, it is the product of the 40 PatpB-601 NEP promoter, with readily detectable activity only in white iojap leaves.

- 20 5 Sequence conservation around the monocot NEP transcription initiation sites. 10 Alignment of the maize clpP, rpoB and atpB NEP promoters revealed significant homology upstream of the transcription initiation sites. In a 13-nucleotide region 8 nucleotides are shared in all three promoters (Fig. 5; Box I). In a pairwise comparison, atpB/clpP, atpB/rpoB 15 and clpP/rpoB share 11, 10 and 8 nucleotides, respectively. In addition, atpB and clpP share 8 out of 9 nucleotides further upstream (-21 to -30 relative to transcription initiation site; Box II in Fig. 5). Interestingly, each maize NEP promoter has 20 sequence similarity around the transcription initiation site with the loose dicot NEP promoter consensus CATAGAATA/GAA (Hajdukiewicz et al., 1997, supra; underlined in Fig. 5). For clpP, 9 nucleotides are conserved out of 10; for atpB and rpoB, the number of 25 conserved nucleotides is 7 out of 10 (Fig. 5). In addition, a second conserved region (Box II, Figure 5) is found upstream of Box I in atpB and clpP, but not in the rpoB promoters. Interestingly, the moncot Box II contains truncated versions of the dicot NEP consensus in 30 a direct orientation: 7 out of 10 bp match in case of the maize clpP (ATAGAAT) and atpB (AT-GAATA) genes (Fig. 5). These tandem repeats may play a role in regulating NEP promoter activity. The regions containing the tentative maize NEP 35 promoter sequences (-30 to +25) have been aligned with homologous regions from other monocot plants. See Figure 5B-5D. The high degree of sequence conservation indicates the presence of functional promoters in each of these monocots species. Promoter activity for the rice 40 homologue of the maize PclpP-111 promoter has been confirmed by primer extension analysis (data not shown).

- 21 5 The data reported herein show that maize plastid NEP promoter regions share sequence homology around the transcription initiation site with the conserved CATAGAATA/GAA NEP sequence motif in tobacco (Fig. 5). Therefore, these promoters are considered to 10 be Type I NEP promoters. This finding indicates conservation of the NEP transcription machinery between monocots and dicots. Sequences upstream of the transcription initiation sites are conserved more extensively than downstream sequences among the maize 15 clpP, rpoB, and atpB promoters, as shown in Figure 5, similar to the dicot Type I NEP promoters (Hajdukiewicz et al., 1997, supra). Both, the maize PclpP-lll promoter and the tobacco PclpP-53 promoter are constitutive. In contrast 20 to the maize promoter, the clpP promoter region in tobacco lacks the CATAGAATA/GAA sequence motif (Hajdukiewicz et al., 1997, supra) suggesting recognition by a different NEP specificity factor (Type II NEP promoter). Interestingly, in Type II NEP promoters 25 sequences downstream of the transcription initiation sites are conserved more extensively than upstream as described in the following example. The tobacco PclpP-53 promoter homologues are the only known examples for plastid Type II NEP promoters. They have been highly 30 conserved during evolution, including the liverworth Machantia polymorpha and the conifer Pinus contorta. Although DNA sequences required for clpP Type II NEP promoter function are maintained, this region is transcriptionally silent in maize, rice and wheat. Lack 35 of transcription from this region in cereals is probably due to the loss of Type II recognition specificity. See Example II. Accordingly, the tobacco (dicot) Type II clpP promoter is suitable to drive the expression of plastid transgenes only in dicots, whereas the cereal 40 Type I promoter may be useful in both monocots and dicots.

- 22 5 The rpoB operon is one of few genes known to be expressed by NEP only. The PrpoB-147 is a Type I NEP promoter but, unlike clpP and atpB, lacks Box II (Fig. 5). Accumulation of mRNA from this promoter is low in mature leaves due to down-regulation of transcription 10 rates (Baumgartner et al., Plant Physiology 101:781-791, 1993). The PrpoB -147 promoter identified in this study probably plays a central role in plastid development since it regulates expression of four out of the five plastid-encoded PEP subunits (Shimada et al., Mol. Gen. 15 Genet. 221:395-402, 1990). According to one model, the two RNA polymerases form a developmental cascade during chloroplast differentiation. During the early stages of plastid development, plastid genes encoding the plastid's transcription and translation apparatus would be 20 transcribed NEP. Once PEP is made, it would initiate transcription of photosynthetic genes from PEP promoters, and take over transcription of housekeeping genes from alternative PEP promoters (Hess et al., 1993, supra; Lerbs-Mache, Proc. Natl. Acad. Sci. 90:5509-5513, 1993; 25 Mullet, Plant Physiol. 103:309-313, 1993; Hajdukiewicz et al. 1997, supra). Consistent with this model is transcription of rpoB from a NEP promoter. However, in maize at least one gene, clpP, is exclusively and efficiently transcribed by NEP in mature chloroplasts 30 indicating that NEP remains active and essential for cellular functions even if PEP is present. An alternative hypothesis is proposed herein, which assumes that NEP and PEP are constitutively present all the time and selective transcription is mediated by promoter-specific 35 transcription factors. The identification of NEP promoters for dicots (Hajdukiewicz et al., 1997, supra) and monocots (described herein) facilitates the elucidation of the roles these two plastid RNA polymerases play in plastid function and development. 40 - 23 5 EXAMPLE II Altered clpP Promoter Recognition by the Nucleus-Encoded Plastid RNA Polymerase Suggests Loss of a Conserved Plastid Transcription Factor in Monocots 10 The plastid clpP gene is transcribed by the nuclear-encoded plastid RNA polymerase (NEP) in rice, a monocot, and tobacco, a dicot. However, the two NEP promoters do not share sequence homology. To assess conservation of NEP promoter recognition between monocots 15 and dicots, a reporter gene (uidA) expressed from the rice clpP promoter region has been introduced into tobacco plastids. The data indicate that in tobacco, transcription initiates at the correct site from the rice clpP promoter. Thus, NEP promoter recognition for this 20 gene is conserved in both monocots and dicots. Surprisingly, transcription from the rice sequence initiated at a second site, which possesses a short stretch of homologous sequence similar to the tobacco clpP promoter region. Sequences around the clpP 25 transcription initiation site are conserved in tobacco, a dicot, Marchantia polymorpha, a bryophyte, and Pinus contorta, a conifer. Lack of transcription from this region in rice and other cereals indicates the evolutionary loss of a-factor required for NEP Class II 30 promoter specificity. Materials and Methods for Example II Construction of plasmid pDS44. Plasmid pDS44 is a pLAA24 derivative (Zoubenko 35 et al., Nucleic Acids Res. 22:3819-3824, 1994) which carries a uidA reporter gene expressed from a Prrn promoter. Plasmid pDS44 was obtained by excising the Prrn promoter as an SacI/EcoRI fragment and replacing it with the rice clpP promoter region engineered as a 40 SacI/EcoRI fragment. The 251 nucleotide SacI/EcoRI DNA fragment containing the rice clpP promoter region (including 19 basepairs of the coding region) was obtained by PCR amplification. The sequence of the PCR - 24 5 primers, and the position of their first nucleotide (or of its complement) in the rice plastid genome (Hiratsuka et al., Mol. Gen. Genet. 217:185-194, 1989; GeneBank Accession No. X15901) are: P1 68520(C) qqaqcTCGAATCACCATTCTTT SEQ ID NO: 50 10 P2 68270 gggaattcTTGCAACACCAATGGGCAT SEQ ID NO: 51 Nucleotides derived from the plastid genome are in capital letters; those included to create a restriction site are in lower case letters. SacI or EcoRI restriction sites are underlined. 15 Tobacco plastid transformation. Plastid transformation and regeneration of transgenic tobacco plants was carried out according to the protocol described by Svab and Maliga (Proc. Natl. Acad. Sci. 20 90:913-917, 1993). Briefly, tobacco leaves were bombarded with tungsten particles coated with plasmid pDS44 DNA using the DuPont PDS 100OHe Biolistic gun. Transgenic shoots were selected on RMOP medium containing 500 pg/ml spectinomycin dihydrocloride. Putative primary 25 transformants were identified by histochemical staining for -glucuronidase activity encoded by uidA (Jefferson, In Genetic Engineering, Vol. 10, Settlow, J.K., ed. Plenum Press, NY & London, pp 247-263, 1988). A uniform population of transformed plastid genomes was verified by 30 Southern analysis (data not shown). Primer extension analysis to map RNA 5'-ends. Total leaf RNA was isolated from leaves of in vitro grown plants by the method of Stiekema et al., 35 supra. Primer extension reactions were carried out on 20 pg of RNA with primer uidA PE1 as described by Allison and Maliga (1995) using primer P3: 5'-GGCCGTCGAGTTTT TTGATTTCACGGGTTGGGG-3' (SEQ ID NO:52) (which is complementary to the uidA coding region. 40 DNA Sequence Analysis. DNA sequence analysis - 25 5 was carried out utilizing the Wisconsin Sequence Analysis Package (Genetics Computer Group, Inc.) as described in Example IV. Results and Discussion 10 Construction of Transgenic Plants. The sequence of rice clpP upstream region included as a promoter fragment in plasmid pDS44 is shown in Figure 6. In rice, this region contains the Os-PclpP-111 Type I NEP promoter. The cognate sequence in tobacco contains two 15 NEP promoters and one PEP promoter. The Os-PclpP-111 NEP promoter was cloned upstream of a uidA coding region (encoding 0-glucuronidase or GUS) with a ribosome binding site, and the rps16 3'-untranslated region (Trpsl6) for stabilization of the mRNA shown in Figure 7. The 20 chimeric uidA gene was cloned next to a selectable spectinomcyin resistance (aadA) gene in the pPRV111A plastid vector, Genebank Accession No. U12812, and introduced into tobacco plastids. A schematic drawing of the vector is shown in Figure 7. Plastid transformants 25 were selected on spectinomycin medium. Out of 25 spectinomcyin resistant lines, 12 were GUS positive. Integration of uidA at the target site was confirmed by Southern analysis (data not shown). Primer extension analysis to test promoter 30 activity. Primer extension analysis was carried out to map uidA 5'-ends initiating in the Os-PclpP-111 promoter region. Three major and two minor uidA transcripts were identified in the leaves of transgenic plants (Figure 8A). A major transcript 5'end mapped to nucleotide 35 position -111, the same position as in rice. This result indicates that the rice Type I PclpP-111 promoter is properly recognized in tobacco, a dicot, indicating the broad applicability of this promoter in a variety of plant species. The second major transcript with the most 40 intense signal was found at the rice -61 position. This transcript 5'-end mapped to a rice sequence with a 29 - 26 5 nucleotide stretch of homology to the tobacco Nt-PclpP-53 promoter region (Figure 6). This was unexpected, since this region in rice and maize is transcriptionally silent (Example I). The third major transcript mapped to position -136 which falls within the upstream monocot NEP 10 box (Example I). The two minor transcript 5'-ends mapped to nucleotide positions -169 and -177 (Figure 8). These transcripts are absent in rice leaf RNA, and they do not correspond to any of the known tobacco clpP transcripts. 15 Therefore, these 5'-ends may be primary transcripts of fortuitous PEP or NEP promoters, or are processed mRNA 5'-ends. Sequence alignment of the regions containing 20 the clpP promoters The rice clpP promoter fragment was transcriptionally active in tobacco. This promoter sequence corresponds to the tobacco Type II PclpP-53 promoter. In this region, rice and tobacco share a 29 nucleotide stretch of homologous sequence with 23 25 conserved nucleotides. To test the conservation of this same region during evolution, sequences around the clpP transcription initiation sites were aligned, including those of the liverwort Marchantia polymorpha (Kohchi et al., Curr. Genet. 14:147-154, 1988), the conifer Pinus 30 contorta, (Clarke et al., Plant Mol. Biol. 26:851-862, 1994), dicots tobacco (Hajdukiewicz et al., 1997, supra) spinach, Arabidopsis (Westhoff, Mol. Gen. Genet. 201:115 123, 1985) and the monocots maize and rice. See Figure 9. With the exception of tobacco, a single transcription 35 initiation site was mapped for each of the clpP genes. We have found, that the 29-nucleotide segment around the clpP transcription initiation site is conserved, (underlining) suggesting that clpP is transcribed by NEP in all of these species. 40 The data presented in this Example show that the rice Os-PclpP-111 NEP promoter is properly recognized - 27 5 in tobacco plastids. Transcription from the rice PclpP-111 NEP promoter in tobacco was observed presumably because the region around the transcription initiation site includes the dicot Type I NEP promoter consensus. As in rice, the Os-PclpP-lll NEP promoter is active in 10 mature tobacco leaves. Therefore, it belongs to the relatively small number of Type I NEP promoters which are active in mature chloroplasts as well as proplastids. Transcription from the rice clpP 5' region at a second site, with a short stretch of homologous sequence 15 to the tobacco PclpP -53 Type II NEP promoter was unexpected. Since rice contains all the cis elements required for Type II promoter activity, lack of transcription in rice should be due to the evolutionary loss of the specificity factor required for Type II 20 promoter recognition. This specificity factor is well conserved during evolution, as evidenced by an active Type II clpP promoter in the liverwort Marchantia polymorpha and the conifer Pinus contorta (Figure 9). Experiments reported here for the rice 25 Os-PclpP-lll NEP promoter suggest that the Type I NEP transcription machinery is sufficiently conserved between monocots and dicots to ensure faithful recognition of heterologous promoters. The clpP mRNA accumulates to significant levels in all plastid types (Shanklin et al., 30 1995, supra; Hajdukiewicz et al., 1997, supra; Example I). Therefore, the strong, constitutive Os-PclpP-111 NEP promoter is suitable for the expression of chimeric genes in a broad range of crops. 35 EXAMPLE III In vivo definition of a Type II promoter, PclpP-53, for the nuclear encoded plastid RNA polymerase (NEP) 40 In tobacco the clpP gene is transcribed from three major NEP promoters initiating transcription -511, -173 and -53 nucleotides upstream of the translation initiation codon, and from a PEP promoter (5' end at - 28 5 -95). Transcription from the Type II PclpP-53 NEP promoter is maintained in the green leaves of wild-type tobacco plants. Therefore, given its potential to drive the expression of selectable marker genes, the constitutive PclpP-53 promoter was chosen for analysis. 10 Materials and Methods for Example III Construction of Plasmids. Plasmid pPS8 contains a uidA reporter gene as a SacI-HindIII fragment in a pBSKS+ plasmid (Stratagene). The chimeric uidA gene 15 consists of: Between the SacI and XhoI sites, the PclpP-53(-22/+25) promoter fragment containing 22 nt upstream and 25 nt downstream (+1 is nt where transcription initiates) of the clpP transcription initiation site; Between XhoI and NcoI sites, a ribosome 20 binding site; Between the NcoI and XbaI sites, the uidA coding region with an N-terminal c-myc tag corresponding to amino acids 410-419 (EQKLISEEDL; SEQ ID NO: 53) within the carboxy terminal domain of the human c-myc protein (Kolodziej and Young, Meth. Enz. 194:508-519, 1991); 25 Between the XbaI and HindIII sites the 3' untranslated region of the rps16 ribosomal protein gene (Trps16). DNA sequence of the chimeric uidA gene between the SacI and HindIII sites in plasmid pPS8 is shown in Figure 13. Relevant restriction sites of the chimeric uidA gene are 30 shown in Fig. 10, where the uidA gene is shown as part of plasmid pPS18. Plasmid pPS18 was obtained by cloning the uidA gene as a SacI-HindIII fragment into SacI-HindIII-digested pPRV111A plastid transformation vector. Plasmid pPRV111A, a pBSKS+ plasmid 35 derivative (Strategene), and was described in Zoubenko et al., 1994, supra. Plasmids pPS16, pPS37, pPS17 and pPS38 listed in Figure 11 were obtained from plasmid pPS18 by replacing the PclpP-53(-22/+2 5 ) SacI-XhoI promoter 40 fragment with the PclpP-53(-152/+154), PclpP-53(-152/+41), PclpP-53(-152/+10), - 29 5 PclpP-53(-39/+154) promoters, respectively. The SacI-XhoI fragments were obtained by PCR amplification. PCR primers are listed according to the position of the terminal nucleotide relative to the transcription initiation site (it is the complement of nt 74557 in the tobacco plastid 10 genome: accession no. Z00044): clpP-152 ccgagctcGAATGAGTccATACTTAT SEQ ID NO: 54 cipP-39 ccgagctcAAAACCAATATGAATATTATA SEQ ID NO: 55 clpP -22 ccgagctcTATAAAGAcAATAAAAAAAAT SEQ ID NO: 56 clpP+10 ccctcgaGAAACGTAACAATTTTTTTT SEQ ID NO: 57 15 clpP+25 ccctcgagTTTCACTTTGAGGTGGA SEQ ID NO: 58 clpP+4. ccctcgagAGAACTAAATACTATATTTC SEQ ID NO: 59 clpP+154 ccctcgagATATGACCCAATATATCTG SEQ ID NO: 60 Anchor sequences derived from the plastid genome are in capital letters ; added nucleotides to 20 create restriction sites (underlined) are in lower case letters. Tobacco Plastid Transformation. Plastid 25 transformation and regeneration of tobacco plants was carried out as described by Svab and Maliga, 1993, supra. Transgenic plants were selected on regeneration medium containing 500 pg/ml spectinomycin dihydrochloride. 30 Primer Extension Analysis. Total leaf RNA was isolated from the leaves of transgenic plants maintained on P.M medium, by the method of Stiekema et al.,1988, supra. Primer extension reactions were carried out as described by Allison and Maliga, 1995, supra, using 15 jig 35 of the total RNA and primer PEl complimentary to the 5' end of the uidA coding sequence. Primer PEl sequence: 5I-GGCCGTCGAGTTTTTTGATTTCACGGGTTGGGG-3' (SEQ ID NO: 61) Results and Discussion 40 To identify functionally important sequences in the Type II PclpP-53 promoter, expression of reporter genes driven by sequences surrounding the clpP-53 NEP - 30 5 transcription initiation site was measured. Deletion of sequences from the 5' and 3' ends facilitated the determination of the boundaries of the promoter. These studies revealed that a 47-bp fragment is sufficient to support accurate transcription initiation. The data 10 further suggest that not more than 28 bp out of 47 are essential for promoter function. A majority of the relevant sequences are downstream of the transcription initiation site. Since all plastid promoters are within about 15 150 nucleotides of the transcription initiation site, transcription initiation in vivo from a 306-bp fragment (-152/+154) surrounding the clpP -53 transcription initiation site was assessed. For testing promoter function, clpP fragments were cloned upstream of a uidA 20 reporter construct encoding 0-glucuronidase. See Figure 11. The uidA construct has a ribosome binding site between the XhoI and NcoI restriction sites, as well as the 3'-untranslated region of the plastid rps16 gene (Trps16) for stabilization of the mRNA. The chimeric 25 uidA gene was introduced into tobacco plastids by linkage to a selectable spectinomycin resistance (aadA) gene. The 306-bp fragment contains a PEP and a NEP promoter (Fig. 11). Functioning of both promoters was established by primer extension analysis. See Figure 12. 30 After confirming that the 306-bp fragment is sufficient to drive NEP transcription, a series of deletions were made from the 5' as well as 3' end. These constructs were then tested for transcription initiation in vivo in tobacco. The schematic design of the promoter 35 deletions is shown in Fig. 11, the primer extension data are shown in Fig. 12. Primer extension analysis on the series of deletions showed that sequences extending from -10 to +25 (pPS43; Fig. 12) and -5 to +25 (pPS44; not shown) are sufficient to support accurate initiation from 40 clpP -53. Upon overexposure, a faint band or the proper size is observed even in the +1 to +25 construct (pPS45, - 31 5 not shown). Also, transcription from the NEP promoter was abolished completely in the -152/+10 construct, (pPS17; Fig. 12) and in the -22 to +21 construct, (pPS41; not shown). This indicates that sequences between +5 and +25 are important for transcription from the NEP 10 promoter. Some of this data is summarized in Figs. 11 and 12. Sequences required for transcription by the NEP polymerase are not known. Based on conservation of the ATAGAATA/GAA around the transcription initiation site, 15 Hajdukiewicz et al., 1997, supra, most NEP promoters were classified as Type I. The promoter studied here, PclpP-53, lacks this sequence motif, because of which it is classified as Type II. Transcription analysis in the truncated promoter fragment of plasmid pPS18 in vivo 20 shows that sequences required to support transcription from PclpP-53 are located within a 30 basepair fragment extending from -5 to +25 with respect to the transcription initiation site. Furthermore, nucleotides between +10 and +25 are important for transcription 25 initiation, since there is no transcription from clpP promoter derivatives in plasmid pPS17 and pPS41 lacking this region. Expression of the rice clpP promoter region revealed a transcript that mapped to a region with 30 homology to the transcription initiation site of the PclpP-53 promoter. Alignment of the rice and the tobacco sequences show significant homology downstream of the transcription initiation site, and not much homology upstream. considering that this rice sequence was 35 recognized by tobacco in vivo (Example II), sequences that are important for transcription initiation should be present in the rice sequence. Combining this information with the finding that -22/+25 sequences are sufficient for transcription initiation in vivo, it appears that 40 sequences 5'-ATTGTTACGTTTCCACCTCAAAGTGAAA-3' (portion of SEQ ID NO: 25) extending from -3 to +25 contain the - 32 5 information which is important for PclpP-53 promoter function. Since the constitutive Type II PclpP-53 is efficiently transcribed in all tissue types, it is useful for the expression of selectable marker genes, and of 10 proteins of economic value in all dicot plants. EXAMPLE IV PLASTID PROMOTER UTILIZATION IN RICE EMBRYOGENIC CELL CULTURE 15 The utilization of the clpP promoter in tobacco and maize plastids is described in the previous examples. The present example is directed to the analysis of plastid promoter utilization in rice. The 5' ends of 20 several mRNA species were mapped in samples derived from cultured embryogenic rice cells and leaves. The RNAs for clpP and 16SrRNA are relatively abundant in embryogenic rice cells indicating that the promoters of these genes may be used to advantage to drive plastid 25 expression of selectable marker genes and/or foreign genes of interest in rice. Plastid transformation in rice is highly desirable. The present example provides compositions and methods to effectuate rice plastid transformation in 30 embryogenic cultured rice cells. Such cells may be efficiently regenerated into mature plants (Vasil, IK (1994) Plant Mol Biol 25:925-937; Christou, P (1996) Trends Plant Sci 1:423-431). Data from cultured tobacco (BY2) cells suggest that plastid promoter utilization in 35 tissue culture may be different from those in leaves (Vera A, Sugiura M (1995) Curr Genet 27:280-284; Vera A, Hirose T, Sugiura M (1996) Mol Gen Genet 251:518-525 et al., 1996; Kapoor S, Suzuki JY, Sugiura M (1997) Plant J 11:327-337). The data presented herein reveal 40 that cultured embryogenic rice cells and leaves utilize the same promoter. rbcL, atpB, 16SrDNA and clpP have only one promoter each which is recognized by the - 33 5 plastid-encoded plastid RNA polymerase (PEP). In contrast, clpP is transcribed by the nucleus-encoded plastid RNA polymerase (NEP) in both samples. The RNAs for clpP and 16SrRNA are relatively abundant in embryogenic cells indicating that the promoters of these 10 genes may be suitable to drive the expression of selectable marker genes. Materials and methods for Example IV Plant Materials. Embryogenic rice callus was 15 initiated from mature seed of cv. Taipei 309 on LS2.5 medium (Abdullah R, Cocking EC, Thompson JA (1986) Bio/Technology 4:1088-1090). The calli were introduced into liquid AA medium (Muller AJ, Grafe R (1978) Mol Gen Genet 161: 67-76) to establish embryogenic suspension 20 cultures, and subcultured at biweekly intervals. DNA and RNA were prepared from 3 month old cultures 14 days after subculture. Plants were regenerated from embryogenic calli on complete MS medium supplemented with 2 mg/L BAP and 3% sucrose (Murashige T, Skoog F (1962) Physiol Plant 25 15: 473-497 1962) and transferred onto hormone-free MS medium. Leaves for the isolation of nucleic acids were taken from these plants after four months. RNA and DNA gel blots. Total cellular RNA was 30 prepared according to Stiekema et al., 1988, supra. The RNA (5 pg per lane) was subjected to electrophoresis in a formaldehyde-agarose gel, blotted and hybridized (Hajdukiewicz PTJ, Allison LA, Maliga P (1997) EMBO J 16:4041-4048). Double-stranded ptDNA probes were 35 prepared by random-primed 32 P-labeling of PCR-generated or gel-purified DNA fragments. The sequence of the primers used for PCR, along with their positions within the tobacco (N.t.; accession no. Z00044; Shinozaki et al. 1986, supra) or maize (Z.m.; accession no. X86563; Maier 40 et al. 1995, supra) ptDNA are as follows: - 34 5 Gene 5' nt position Sequence SEQ ID NO: in plastid DNA atpB (Z.m.) 55860(C) GAGAGGAATGCAAGTCATTGACA (62) 10 55103 GAGCAGGGTCCGTCAAATC (63) clpP (Z.m.) 69840 ATCCTAGCGTGAGGGAATGCTA (64) 70064(C) AGGTCTGATGGTATATCTCAGTAT (65) The following ptDNA fragments were used as 15 probes: rbcL (N.t.), a BamHI fragment (nucleotides 58047 to 59285 in ptDNA); 16SrDNA (N.t.), EcoRI to EcoRV fragment (nucleotides 138447 to 140855). The probe for tobacco 25S rRNA was from plasmid pKDR1 (Dempsey et al., 1993, supra) containing a 3.75 kb 20 EcoRI fragment from a tobacco 25S/18S locus cloned in plasmid pBR325. Total leaf DNA for relative plastid genome copy number determination was prepared (Mettler IJ (1987) Plant Mol Biol Rep 5:346-349), digested with the EcoRI 25 restriction endonuclease, separated on 0.7% agarose gels, blotted and hybridized with the plastid 16SrDNA and cytoplasmic 25SrDNA probes (Allison et al., 1996, supra). 30 Primer extension analysis. Primer extension reactions were carried out on total leaf RNA as described (Allison and Maliga, 1995, supra). The primers are listed below, with nucleotide position in the published rice plastid genome sequence (Hiratsuka et al., 1989, supra). 35 Gene 5' nt position sequence SEQ ID NO: in plastid DNA rbcL 54124 (C) ACTTGCTTTAGTTTCTGTTTGTGGTGACAT (66) 40 atpB 53287 AGAAGTAGTAGGATTGGTTCTCATAAT (67) 16S rRNA 123777 CCGCCAGCGTTCATCCTGAGC (68) clpP 68263 GGTACTTTTGGAACACCAATCGGCAT (69) Primer extension reactions were carried out with 1 pg of 45 RNA from leaves, and 10 yg (clpP, 16SrDNA) and 30 sg (rbcL, atpB) of RNA from embryogenic cells. Results and Discussion Several plastid promoters have been identified - 35 5 in rice and related monocots. To assess whether these promoters would be suitable to drive expression of selectable marker genes and/or foreign genes of interest in these plant species, transcript accumulation was examined. The rbcL gene in rice and maize is transcribed 10 from a PEP promoter (Mullet et al., 1985, supra; Nishiziwa Y, Hirai A (1987) Jpn J Genet 62:389-395). The atpB gene in maize chloroplasts is transcribed from a PEP promoter (Mullet et al., 1985, supra), whereas in maternal white iojap seedlings lacking PEP it is 15 transcribed from an alternative NEP promoter. The 16SrDNA gene (the first gene of the plastid ribosomal RNA operon) in barley chloroplasts is transcribed from a PEP promoter (Reinbothe S, Reibothe C, Heintzen C, Seidenbecher C, Parthier B (1993) EMBO J 12:1505-1512), 20 whereas in the white albostrians seedlings lacking PEP it is transcribed from an uncharacterized NEP promoter (Hess et al., 1993, supra). The clpP gene in wild-type and iojap maize chloroplasts is transcribed from a NEP promoter. 25 To determine the level of expression of these genes in embryogenic rice cells and leaf cells, accumulation of mRNAs was assessed on Northern blots. The data reveal that transcript levels in embryogenic cells relative to leaves were barely detectable for rbcL 30 (153-times lower), reduced for atpB (37-fold lower) and 16SrDNA (7-fold lower), and similar for clpP (approximately 1.1-times lower). See Figure 14. Interestingly, the number of plastid genome copies (ptDNA) per cell is about the same in embryogenic cells 35 and leaves. See Figure 15. Accordingly, the differences in transcript levels represent values normalized for ptDNA copy number. To further characterize active plastid promoters, transcript 5' ends were mapped by primer 40 extension in cultured embryogenic cells and in leaves. Two different 5' ends, at 312 and 58 nucleotides upstream - 36 5 of the translation initiation codon, were mapped for rbcL. See Figure 16. The same two 5'-ends were identified in leaf chloroplasts, and in the plastids of embryogenic cells. Two 5'-ends were mapped to similar positions by Si nuclease analysis in rice chloroplasts 10 (Nishizawa and Hirai, 1987, supra). The rbcL -312-end is downstream of -10/-35 a7-type promoter elements which are conserved in monocots; the -58-end is generated by RNA processing (Mullet et al., 1985, supra; Reinbothe et al., 1993, supra). 15 For atpB, a single 5'-end was mapped 310 nucleotides upstream of the translation initiation codon. As for rbcL, the same 5'-ends were identified in embryogenic cells and leaves. See Figure 16. The -310 end is associated with PEP promoter elements, and has 20 been reported earlier in chloroplasts for rice (Nishizawa Y, Hirai A (1989) Jpn J Genet 64:223-229) and maize (Mullet et al., 1985, supra). For the rRNA operon, the same two 5' ends were mapped upstream of the mature 16SrRNA in embryogenic 25 cells and in leaves shown in Figure 16. Based on DNA sequence conservation, the -116 end is the product of a PEP promoter whereas the -28 end derives from RNA processing. See Figure 17A; Strittmatter G, Godzicka-Josefiak A, Kossel H (1985) EMBO J 4:599-604; 30 Vera and Sugiura, 1995, supra; Allison et al., 1996, supra). For clpP, the same 5' end was mapped in embryogenic tissue culture cells and in leaves. See Figure 16. The transcript initiates 111 nucleotides 35 upstream of the translation initiation codon within the 10-nucleotide NEP consensus, shown in Figure 5A, and is the product of the clpP Type-I NEP promoter. Mapping of RNA 5' ends upstream of rbcL, atpB, 16SrDNA and clpP identified the same promoters in 40 cultured embryogenic cells and in the leaves of rice. The data obtained from these studies in rice can be - 37 5 contrasted with the results reported for tobacco. In tobacco, atpB and 16SrDNA are preferentially transcribed from PEP promoters in leaves, and from NEP promoters in BY2 tissue culture cells (Vera and Sugiura 1995; Kapoor et al. 1997). In rice embryogenic cultures, no PEP to 10 NEP promoter switch was observed for any of the plastid genes examined. The data suggest that a PEP to NEP promoter switch is not essential for adaptation to cell culture. Alternatively, atpB and 16SrDNA, genes which have PEP and NEP promoters in other monocots as discussed 15 in the previous examples, have no NEP promoters in rice. One important difference between embryogenic rice cells and the tobacco BY2 cell line is the length of time in culture. The rice cell culture line described herein is only a few month old and has maintained the 20 ability to regenerate plants. The BY2 cell line has been grown in culture for several years and has lost the capacity for plant regeneration (Yasuda T, Kuroiwa T, Nagata T (1988) Planta 174:235-241). Accordingly the possibility remains that in BY2 cells plastid gene 25 expression may be different from tobacco cells in a short-term culture or in a tobacco plant. The results presented herein provide practical implications for the production of transgenic rice cells and or plants. Two powerful promoters are described 30 herein which are active in rice embryogenic cells: the clpP NEP promoter and the rrn PEP promoter. Both promoters are suitable to drive the expression of selectable marker genes for plastid transformation in embryogenic rice cells. 35 While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from 40 the scope and spirit of the present invention, as set forth in the following claims.

Claims (14)

1. An isolated rpo B promoter element and homologues thereof when used for enhancing production of at least one exogenous protein of interest in plastids of plant cells, selected from the group of promoter elements encoded by SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 11.

2. An isolated atpB promoter element and homologues thereof when used for enhancing production of at least one exogenous protein of interest in plastids of plant cells, selected from the group of promoter elements encoded by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.

3. An isolated clpP promoter element and homologues thereof when used for enhancing production of at least one exogenous protein of interest in plastids of plant cells, selected from the group of promoter elements encoded by SEQ ID NO: 3, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17. SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 30 and SEQ ID NO: 31.

4. An isolated 16SrDNA promoter element and homologues thereof when used for enhancing production of at least one exogenous protein of interest in the plastids of plant cells, selected from the group of promoter elements encoded by SEQ ID NO: 28 and SEQ ID NO: 29. -55

5. A DNA construct for stably transforming the plastids of higher plants, comprising: a) a transcription unit encoding at least one exogenous protein of interest; b) a first NEP promoter being cipP-111 and a second PEP promoter being Prrn-114 in tandem, operably linked to said transcription unit; and c) expression of said transcription unit being regulated by said promoters.

6. A DNA construct for stably transforming the plastids of higher plants, comprising: a) a transcription unit encoding at least one exogenous protein of interest; b) a first NE? promoter being clpP-53 and a second PEP promoter being Prrn-114 in tandem, operably linked to said transcription unit; and c) expression of said transcription unit being regulated by said promoters.

7. Use of a promoter element of any one of claims 1-4 in a plastid transformation vector to enhance the production of at least one exogenous protein in plastids of plant cells.

S. The use according to claim 7, wherein said plant cells are from a non-green tissue.

9. The use according to claim 7, wherein a PEP promoter is used in combination with said promoter element. -56

10. A DNA construct for stably transforming the plastids of higher plants, comprising: a) a transcription unit encoding at least one exogenous protein of interest; b)a rpo B promoter element selected from the group of promoter elements encoded by SEQ ID NO: 1, SEQ ID: 9, and SEQ ID NO: 11, operably linked to said transcriptional unit, wherein expression of said transcription unit is regulated by said promoters.

11. A DNA construct for stably transforming the plastids of higher plants, comprising: a) a transcription unit encoding at least one exogenous protein of interest; b) an atpB promoter element selected from the group of promoter elements encoded by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 operably linked to said transcriptional unit, wherein expression of said transcription unit is regulated by said promoters.

12. A DNA construct for stably transforming the plastids of higher plants, comprising: a) a transcription unit encoding at least one exogenous protein of interest; b) a clpP promoter element selected from the group of promoter elements encoded by SEQ ID NO: 3, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 30 and SEQ ID NO: 31 operably linked to said transcriptional unit, wherein expression of said transcription unit is regulated by said promoters- -57

13. A DNA construct for stably transforming the plastids of higher plants, comprising: a) a transcription unit encoding at least one exogenous protein of interest; b) a 16SrDNA promoter element selected from the group of promoter elements encoded by SEQ ID NO: 28 and SEQ ID NO: 29, operably linked to said transcriptional unit, wherein expression of said transcription unit is regulated by said promoters.

14. The DNA construct of any one of claims 10-14, comprising a PEP promoter.