Chloroplast Transformation Research Articles

High-frequency transformation of undeveloped plastids in tobacco suspension cells.

Although leaf chloroplast transformation technology was developed more than a decade ago, no reports exist of stable transformation of undeveloped plastids or other specialized plastid types, such as proplastids, etioplasts, or amyloplasts. In this work we report development of a dark-grown tobacco suspension cell model system to investigate the transformation potential of undeveloped plastids. Electron microscope analysis confirmed that the suspension cells carry plastids that are significantly smaller (approximately 50-fold less in volume) and have a very different subcellular localization and developmental state than leaf cell chloroplasts. Using antibiotic selection in the light, we demonstrated that both plastid and nuclear transformation of these cell suspensions is efficient and reproducible, with plastid transformation frequency at least equal to that of leaf chloroplast transformation. Homoplasmic plastid transformants are readily obtained in cell colonies, or in regenerated plants, providing a more consistent and versatile model than the leaf transformation system. Because of the uniformity of the cell suspension model, we could further show that growth rate, selection scheme, particle size, and DNA amount influence the frequency of transformation. Our results indicate that the rate-limiting steps for nuclear and plastid transformation are different, and each must be optimized separately. The suspension cell system will be useful as a model for understanding transformation in those plant species that utilize dark-grown embryogenic cultures and for characterizing the steps that lead to homoplasmic plastid transformation.

Substitutions at the Asp-473 Latch Residue of Chlamydomonas Ribulosebisphosphate Carboxylase/Oxygenase Cause Decreases in Carboxylation Efficiency and CO2/O2 Specificity

The loop between alpha-helix 6 and beta-strand 6 in the alpha/beta-barrel active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) plays a key role in discriminating between gaseous substrates CO(2) and O(2). Based on numerous x-ray crystal structures, loop 6 is either closed or open depending on the presence or absence, respectively, of substrate ligands. The carboxyl terminus folds over loop 6 in the closed conformation, prompting speculation that it may trigger or latch loop 6 closure. Because an x-ray crystal structure of tobacco Rubisco revealed that phosphate is located at a site in the open form that is occupied by the carboxyl group of Asp-473 in the closed form, it was proposed that Asp-473 may serve as the latch that holds the carboxyl terminus over loop 6. To assess the essentiality of Asp-473 in catalysis, we used directed mutagenesis and chloroplast transformation of the green alga Chlamydomonas reinhardtii to create D473A and D473E mutant enzymes. The D473A and D473E mutant strains can grow photoautotrophically, indicating that Asp-473 is not essential for catalysis. However, both substitutions caused 87% decreases in carboxylation catalytic efficiency (V(max)/K(m)) and approximately 16% decreases in CO(2)/O(2) specificity. If the carboxyl terminus is required for stabilizing loop 6 in the closed conformation, there must be additional residues at the carboxyl terminus/loop 6 interface that contribute to this mechanism. Considering that substitutions at residue 473 can influence CO(2)/O(2) specificity, further study of interactions between loop 6 and the carboxyl terminus may provide clues for engineering an improved Rubisco.

A simple method for chloroplast transformation in Chlamydomonas reinhardtii.

Photosystem (PS)I is a multi-subunit pigment-protein complex that uses light energy to transfer electrons from plastocyanin to ferredoxin. Application of genetic engineering to photo-synthetic reaction center proteins has led to a significant advancement in our understanding of primary electron transfer events and the role of the protein environment in modulating these processes. Chlamydomonas reinhardtii provides a system particularly amenable to analyze the structure-function relationship of PSI. Chlamydomonas reinhardtii is also a favorable organism for chloroplast transformation because it contains a single chloroplast and grows heterotrophically when supplemented with acetate. Chlamydomonas has served as a model organism for the development of chloroplast transformation procedures and the study of photosynthetic mutants generated using this method. Exogenous cloned cpDNA can be introduced into the chloroplast by using this biolistic gene gun method. DNA-coated tungsten or gold particles are bombarded onto cells. Upon its entry into chloroplasts, the transforming DNA is released from the particles and integrated into the chloroplast genome through homologous recombination. The most versatile chloroplast selectable marker is aminoglycoside adenyl transferase (aadA), which can be expressed in the chloroplast to confer resistance to spectinomycin or streptomycin. This chapter describes the procedures for chloroplast transformation.

Engineering the Chloroplast Encoded Proteins of Chlamydomonas

Over a decade ago (1988), John Boynton and colleagues successfully transformed the chloroplast genome of chlamydomonas for the first time by complementation of a chloroplast deletion mutant. Since the first demonstration of chloroplast transformation the function and structure of many chloroplast encoded subunits of the photosynthetic apparatus has been characterized by site-directed mutagenesis. With the completion of the sequencing of the Chlamydomonas chloroplast genome the genetic tools are now in hand to characterize structure-function relationships for each of the chloroplast-encoded proteins of the photosynthetic apparatus.

Plastid protein synthesis is required for plant development in tobacco.

Chloroplasts fulfill important functions in cellular metabolism. The majority of plastid genome-encoded genes is involved in either photosynthesis or chloroplast gene expression. Whether or not plastid genes also can determine extraplastidic functions has remained controversial. We demonstrate here an essential role of plastid protein synthesis in tobacco leaf development. By using chloroplast transformation, we have developed an experimental system that produces recombination-based knockouts of chloroplast translation in a cell-line-specific manner. The resulting plants are chimeric and, in the presence of translational inhibitors, exhibit severe developmental abnormalities. In the absence of active plastid protein synthesis, leaf blade development is abolished because of an apparent arrest of cell division. This effect appears to be cell-autonomous in that adjacent sectors of cells with translating plastids are phenotypically normal but cannot complement for the absence of plastid translation in mutant sectors. Developmental abnormalities also are seen in flower morphology, indicating that the defects are not caused by inhibited expression of plastid photosynthesis genes. Taken together, our data point to an unexpected essential role of plastid genes and gene expression in plant development and cell division.

Assessment of Structural and Functional Divergence Far from the Large Subunit Active Site of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

Despite conservation of three-dimensional structure and active-site residues, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) enzymes from divergent species differ with respect to catalytic efficiency and CO2/O2 specificity. A deeper understanding of the structural basis for these differences may provide a rationale for engineering an improved enzyme, thereby leading to an increase in photosynthetic CO2 fixation and agricultural productivity. By comparing 500 active-site large subunit sequences from flowering plants with that of the green alga Chlamydomonas reinhardtii, a small number of residues were found to differ in regions previously shown by mutant screening to influence CO2/O2 specificity. When directed mutagenesis and chloroplast transformation were used to change Chlamydomonas Met-42 and Cys-53 to land plant Val-42 and Ala-53 in the large subunit N-terminal domain, little or no change in Rubisco catalytic properties was observed. However, changing Chlamydomonas methyl-Cys-256, Lys-258, and Ile-265 to land plant Phe-256, Arg-258, and Val-265 at the bottom of the alpha/beta-barrel active site caused a 10% decrease in CO2/O2 specificity, largely due to an 85% decrease in carboxylation catalytic efficiency (Vmax/Km). Because land plant Rubisco enzymes have greater CO2/O2 specificity than the Chlamydomonas enzyme, this group of residues must be complemented by other residues that differ between Chlamydomonas and land plants. The Rubisco x-ray crystal structures indicate that these residues may reside in a variable loop of the nuclear-encoded small subunit, more than 20 A away from the active site.

Chlamydomonas reinhardtii at the crossroads of genomics.

Simple, experimentally tractable systems such Saccharomyces cerevisiae, Chlamydomonas reinhardtii, and Arabidopsis thaliana are powerful models for dissecting basic biological processes. The unicellular green alga C. reinhardtii is amenable to a diversity of genetic and molecular manipulations. This haploid organism grows rapidly in axenic cultures, on both solid and liquid medium, with a sexual cycle that can be precisely controlled. Vegetative diploids are readily selected through the use of complementing auxotrophic markers and are useful for analyses of deleterious recessive alleles. These genetic features have permitted the generation and characterization of a wealth of mutants with lesions in structural, metabolic and regulatory genes. Another important feature of C. reinhardtii is that it has the capacity to grow with light as a sole energy source (photoautotrophic growth) or on acetate in the dark (heterotrophically), facilitating detailed examination of genes and proteins critical for photosynthetic or respiratory function. Other important topics being studied using C. reinhardtii, many of which have direct application to elucidation of protein function in animal cells (26), include flagellum structure and assembly, cell wall biogenesis, gametogenesis, mating, phototaxis, and adaptive responses to light and nutrient environments (32, 44). Some of these studies are directly relevant to applied problems in biology, including the production of clean, solar-generated energy in the form of H2, and bioremediation of heavy metal wastes. Recent years have seen the development of a molecular toolkit for C. reinhardtii (42, 44, 66, 98, 99). Selectable markers are available for nuclear and chloroplast transformation (4, 5, 12, 13, 30, 44, 56, 82). The Arg7 (22) and Nit1 (30) genes are routinely used to rescue recessive mutant phenotypes. The bacterial ble gene (which codes for zeocin resistance [70, 112]) is an easily scored marker for nuclear transformation, and the bacterial aadA gene (which codes for spectinomycin and streptomycin resistance) is a reliable marker for chloroplast transformation (39). Nuclear transformation can be achieved by

Expression of the B subunit of E. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization.

Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1–ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.

Low frequency transmission of a plastid-encoded trait in Setaria italica.

It has been claimed that engineering traits into the chloroplast will prevent transgene transmission by pollen, precluding transgene flow from crops. A Setaria italica (foxtail or birdseed millet) with chloroplast-inherited atrazine resistance (bearing a nuclear dominant red-leaf base marker) was crossed with five male-sterile yellow- or green-leafed herbicide susceptible lines. Chloroplast-inherited resistance was consistently pollen transmitted at a 3x10(-4 )frequency in >780,000 hybrid offspring. The nuclear marker segregated in the F(2), but resistance did not segregate, as expected. Pollen transmission of plastome traits can only be detected using both large samples and selectable genetic markers. The risk of pollen transmission at this frequency would be several orders of magnitude greater than spontaneous nuclear-genome mutation-rates. Chloroplast transformation may be an unacceptable means of preventing transgene outflow, unless stacked with additional mechanisms such as mitigating genes and/or male sterility.

Rapid and proven production of transplastomic tobacco plants by restoration of pigmentation and photosynthesis.

Tobacco chloroplast transformation is typically achieved using dominant, selectable antibiotic resistance genes such as aadA, nptII and aphA-6. An improvement would be the combination of such a marker with a visual screening system for the early and conclusive detection of plastid transformants. As such, we investigated the use of three photosynthesis-deficient plastid mutants, DeltapetA, Deltaycf3 and DeltarpoA, for the development of a phenotypic selection system. Mutant plants were used as an alternative to the wild-type as source tissue for transformation, re-introducing deleted plastid sequences and using the aphA-6 gene as a selection marker. The reconstitution of the deleted genes in transformed regenerants resulted in shoots with a visually distinct phenotype comparable to the wild-type. This transformation/selection system overcomes the common problems associated with plastid transformation, e.g. the recovery of spontaneous mutants or nuclear insertions. In addition to the benefits offered by phenotypic selection, phenotype reconstitution leads to restoration of photosynthesis, which we assume drives reconstituted plants rapidly towards homoplasmy. As such, repeated cycles of regeneration in the presence of an antibiotic selection agent are no longer required.

The Relationship between Side Reactions and Slow Inhibition of Ribulose-bisphosphate Carboxylase Revealed by a Loop 6 Mutant of the Tobacco Enzyme

The first directed mutant of a higher plant ribulose-bisphosphate carboxylase/oxygenase (Rubisco), constructed by chloroplast transformation, is catalytically impaired but still able to support the plant's photosynthesis and growth (Whitney, S. M., von Caemmerer, S., Hudson, G. S., and Andrews, T. J. (1999) Plant Physiol. 121, 579-588). This mutant enzyme has a Leu to Val substitution at residue 335 in the flexible loop 6 of the large subunit, which closes over the substrate during catalysis. Its active site was intact, as judged by its barely impaired competency in the initial enolization step of the reaction sequence, and its ability to bind tightly the intermediate analog, 2'-carboxy-D-arabinitol-1,5-bisphosphate. Prompted by observations that the mutant enzyme displayed much less slow inhibition during catalysis in vitro than the wild type, its tendency to catalyze side reactions and its response to the slow inhibitor D-xylulose-1,5-bisphosphate were studied. The lessening in slow inhibition was not caused by reduced production of inhibitory side products. Except for pyruvate production, these reactions were strongly enhanced by the mutation, as was the ability to catalyze the carboxylation of D-xylulose-1,5-bisphosphate. Rather, reduced inhibition was the result of lessened sensitivity to these inhibitors. The slow isomerization phase that characterizes inhibition of the wild-type enzyme by D-xylulose-1,5-bisphosphate was completely eliminated by the mutation, and the mutant was more adept than the wild type in catalyzing the benzylic acid-type rearrangement of D-glycero-2,3-pentodiulose-1,5-bisphosphate (produced by oxidation of the substrate, D-ribulose-1,5-bisphosphate). These observations are consistent with increased flexibility of loop 6 induced by the mutation, and they reveal the underlying mechanisms by which the side reactions cause slow inhibition.

Phytoremediation of organomercurial compounds via chloroplast genetic engineering.

Mercury (Hg), especially in organic form, is a highly toxic pollutant affecting plants, animals, and man. In plants, the primary target of Hg damage is the chloroplast; Hg inhibits electron transport and photosynthesis. In the present study, chloroplast genetic engineering is used for the first time to our knowledge to enhance the capacity of plants for phytoremediation. This was achieved by integrating a native operon containing the merA and merB genes (without any codon modification), which code for mercuric ion reductase (merA) and organomercurial lyase (merB), respectively, into the chloroplast genome in a single transformation event. Stable integration of the merAB operon into the chloroplast genome resulted in high levels of tolerance to the organomercurial compound, phenylmercuric acetate (PMA) when grown in soil containing up to 400 micro M PMA; plant dry weights of the chloroplast transformed lines were significantly higher than those of wild type at 100, 200, and 400 micro M PMA. That the merAB operon was stably integrated into the chloroplast genome was confirmed by polymerase chain reaction and Southern-blot analyses. Northern-blot analyses revealed stable transcripts that were independent of the presence or absence of a 3'-untranslated region downstream of the coding sequence. The merAB dicistron was the more abundant transcript, but less abundant monocistrons were also observed, showing that specific processing occurs between transgenes. The use of chloroplast transformation to enhance Hg phytoremediation is particularly beneficial because it prevents the escape of transgenes via pollen to related weeds or crops and there is no need for codon optimization to improve transgene expression. Chloroplast transformation may also have application to other metals that affect chloroplast function.

The stem-loop region of the tobacco psbA 5'UTR is an important determinant of mRNA stability and translation efficiency.

Regulation of chloroplast gene expression involves networked and concerted interactions of nucleus-encoded factors with their target sites on untranslated regions (UTRs) of chloroplast transcripts. So far, only a few cis-acting elements within such 5'UTR sequences have been identified as functional determinants of mRNA stability and efficient translation in Chlamydomonas in vivo. In this study, we have used chloroplast transformation and site-directed mutagenesis to analyse the functions of the 5'UTRs of tobacco psbA and rbcL fused to the coding region of the reporter gene uidA. Various mutant versions of the psbA leader, as well as rbcL/psbA hybrid leader elements, were investigated. Our results showed a 1.5- to 3-fold decrease in uidA mRNA levels and a 1.5- to 6-fold reduction in uidA translation efficiency in all psbA 5'UTR stem-loop mutants generated by sequence deletions and base alterations. This indicates that the correct primary sequence and secondary structure of the psbA 5'UTR stem-loop are required for mRNA stabilisation and translation. The 5'-terminal segment of the rbcL 5'UTR did not enhance the stability or translational activity of chimeric uidA mRNA under the standard light-dark regime of 16 h light and 8 h dark. Stabilising effects were, however, observed when the cells were kept continuously in the dark. Possible reasons for the influence of the 5'UTR of the tobacco psbA on mRNA stability and translation efficiency are discussed.

Multiple Translational Control Sequences in the 5′ Leader of the Chloroplast psbC mRNA Interact With Nuclear Gene Products in Chlamydomonas reinhardtii

Translation of the chloroplast psbC mRNA in the unicellular eukaryotic alga Chlamydomonas reinhardtii is controlled by interactions between its 547-base 5' untranslated region and the products of the nuclear loci TBC1, TBC2, and possibly TBC3. In this study, a series of site-directed mutations in this region was generated and the ability of these constructs to drive expression of a reporter gene was assayed in chloroplast transformants that are wild type or mutant at these nuclear loci. Two regions located in the middle of the 5' leader and near the initiation codon are important for translation. Other deletions still allow for partial expression of the reporter gene in the wild-type background. Regions with target sites for TBC1 and TBC2 were identified by estimating the residual translation activity in the respective mutant backgrounds. TBC1 targets include mostly the central part of the leader and the translation initiation region whereas the only detected TBC2 targets are in the 3' part. The 5'-most 93 nt of the leader are required for wild-type levels of transcription and/or mRNA stabilization. The results indicate that TBC1 and TBC2 function independently and further support the possibility that TBC1 acts together with TBC3.

Effects of selective inactivation of individual genes for low-molecular-mass subunits on the assembly of photosystem II, as revealed by chloroplast transformation: the psbEFLJoperon in Nicotiana tabacum.

Photosystem (PSII) is a supramolecular polypeptide complex found in oxygenic photosynthetic membranes, which is capable of extracting electrons from water for the reduction of plastoquinone. An intriguing feature of this assembly is the fact that it includes more than a dozen low-mass polypeptides of generally unknown function. Using a transplastomic approach, we have individually disrupted the genes of the psbEFLJoperon in Nicotiana tabacum, which encode four such polypeptides, without impairing expression of downstream loci of the operon. All four mutants exhibited distinct phenotypes; none of them was capable of photoautotrophic growth. All mutants bleached rapidly in the light. Disruption of psbEand psbF, which code for the alpha and beta apoproteins of cytochrome b(559), abolished PSII activity, as expected; Delta psbL and Delta psbJ plants displayed residual PSII activity in young leaves. Controlled partial solubilisation of thylakoid membranes uncovered surprisingly severe impairment of PSII structure, with subunit and assembly patterns varying depending on the mutant considered. In the Delta psbL mutant PSII was assembled primarily in a monomeric form, the homodimeric form was preponderant in Delta psbJ, and, unlike the case in Delta psbZ, the thylakoids of both mutants released some PSII supercomplexes. On the other hand, Photosystem I (PSI), the cytochrome b(6)f complex, ATP synthase, LHCII, and CP24/CP26/CP29 antennae were present in near wild-type levels. The data are discussed in terms of their implications for structural, biogenetic and functional aspects of PSII.

Chloroplast transformation in oilseed rape.

The chloroplast transformation vector pNRAB carries two expression cassettes for the spectinomycin resistance gene aadA and the insect resistance gene cry1Aa10. The two cassettes are sited between the rps7 and ndhB targeting fragments. Biolistic delivery of the vector DNA, followed by spectinomycin selection, yielded chloroplast transformants at a frequency of four in 1000 bombarded cotyledon petioles. PCR analysis and Southern blot of PCR products confirmed the site-specific integration of aadA and cry1Aa10 into the chloroplast genomes of transgenic oilseed rape. When transgenic oilseed rape leaves were fed to second instar Plutella xylostera larvae, 47% mortality was observed against this insect and the surviving larvae had significantly lower weight than the control. This is the first report of chloroplast transformation in oilseed rape and the introduction of novel genes between the rps7 and ndhB genes in the chloroplast genome. This offers an opportunity for improvement of oilseed rape by chloroplast genetic engineering.

History of chloroplast genomics.

The presence of chloroplast DNA was established in 1963. With the development of recombinant DNA technologies, chloroplast DNA was selected as one of the first candidates for genome sequencing. The first physical map was reported for maize chloroplasts in 1976. As tobacco has been popular as an experimental system, tobacco chloroplast DNA has been extensively analyzed and the complete nucleotide sequence was established in 1986. This sequencing and the availability of tobacco chloroplast transformation techniques and of in vitro expression systems have formed the basis of an ongoing functional genomics program.

Complementation of the nuclear antisense rbcS-induced photosynthesis deficiency by introducing an rbcS gene into the tobacco plastid genome.

The small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a nuclear gene-encoded protein that is imported into chloroplasts where it assembles with the large subunit (LS) after removal of the transit peptide to form Rubisco. We have explored the possibility that the severe deficiency in photosynthesis exhibited in nuclear transgenic tobacco (line alpha5) expressing antisense rbcS coding DNA that results in low SS and Rubisco protein content [Rodermel et al. (1988) Cell 55: 673] could be complemented by introducing a copy of the rbcS gene into its plastid genome through chloroplast transformation. Two independent lines of transplastomic plants were generated, in which the tobacco rbcS coding sequence, either with or without the transit sequence, was site-specifically integrated into the plastid genome. We found that compared with the antisense plants, expression of the plastid rbcS gene in the transplastomic plants resulted in very high mRNA abundance but no increased accumulation of the SS and Rubisco protein or improvement in plant growth and photosynthesis. Therefore, there is a limitation in efficient translation of the rbcS mRNA in the plastid or an incorrect processing and modification of the plastid-synthesized SS protein that might cause its rapid degradation.

Room temperature microspectrofluorimetry as a useful tool for studying the assembly of the PSII chlorophyll-protein complexes in single living cells of etiolated Euglena gracilis Klebs during the greening process.

The assembly kinetics of the PSII chlorophyll-protein complexes was followed during the greening of Euglena gracilis by microspectrofluorimetry in vivo, at room temperature, on single living cells. The study was correlated to micro- and submicroscopic events accompanying the proplastid to chloroplast transformation and with the immunolocalization of the LHCPII. Etiolated cells of Euglena gracilis were grown in darkness in Mego's heterotrophic liquid medium under shaking at 25+/-1 degrees C. At the stationary phase of growth, they were exposed to continuous light (330 micromol m(-2) s(-1)) for 72 h. The analyses were carried out on samples collected at different times of illumination. Microspectrofluorimetric data were recorded in the 620-780 nm range (excitation at 436 nm) and were resolved into Gaussian components corresponding to the reaction centres (RCII) and the inner antennae (CP(43-47)) of the PSII and LHCPII. From the RCII/CP(43-47) and LHCPII/PSII ratios, it was inferred that (1) a disconnection between RCII and CP(43-47) syntheses occurs during the lag phase of chloroplast differentiation, RCII being synthesized before the inner antennae. This results in the accumulation of uncoupled PSII Chl-protein complexes; (2) after lag phase, the RCII and CP(43-47) syntheses are connected one to another; (3) the freshly synthesized LHCPII complexes are immediately assembled with the PSII, suggesting that the outer antennae always maintain the form bound to PSII. Micro- and submicroscopical observations and LHCPII immunolocalization were in agreement. These data suggest that microspectrofluorimetry may constitute a useful non-destructive tool for studying the assembly kinetics of PSII, under fully physiological life conditions.

Structural and functional characterization of a transcription-enhancing sequence element in the rbcL gene of the Chlamydomonas chloroplast genome.

The structure and function of a transcription-enhancing sequence element in the coding region of the Chlamydomonas reinhardtii rbcL gene was analyzed in Chlamydomonas chloroplast transformants in vivo. The enhancer sequence is contained within a DNA segment extending from position +108 to position +143, relative to the start site of rbcL gene transcription. The sequence remains functional when inverted or when placed 34 bp closer to or 87 bp further downstream of the basic rbcL promoter. However, it does not function from a site about 250 bp downstream of its original location. Besides promoting transcription initiation from the rbcL promoter, the element is able to augment transcription from the promoter of the Chlamydomonas chloroplast atpB gene, but has an inhibitory effect on transcription from the promoter of the chloroplast ribosomal RNA genes. The results suggest that the enhancer-like sequence acts upon transcription initiation in a position-specific and promoter type-specific manner.