Protein transport into and within chloroplasts.

The chloroplast is a complex organelle which carries out a wide range of metabolic processes such as light capture and the biosynthesis of carbohydrates, fatty acid and amino acids. This organelle consists of three separate membrane systems which enclose three distinct soluble phases. Most of the chloroplast proteins are imported from the cytosol and directed into the six different compartments. This import and intraorganellar sorting process makes the chloroplast an interesting and promising system for the analysis of how proteins interact with and are translocated across biological membranes.

Protein Import into and Sorting inside the Chloroplast Are Independent Processes.

Plastocyanin is a nuclear-encoded chloroplast thylakoid lumen protein that is synthesized in the cytoplasm with a large N-terminal extension (66 amino acids). Transport of plastocyanin involves two steps: import across the chloroplast envelope into the stroma, followed by transfer across the thylakoid membrane into the lumen. During transport the N-terminal extension is removed in two parts by two different processing proteases. In this study we examined the functions of the two cleaved parts, C1 and C2, in the transport pathway of plastocyanin. The results show that C1 mediates import into the chloroplast. C1 is sufficient to direct chloroplast import of mutant proteins that lack C2. It is also sufficient to direct import of a nonplastid protein and can be replaced functionally by the transit peptide of an imported stromal protein. C2 is a prerequisite for intraorganellar routing but is not required for chloroplast import. Deletions in C2 result in accumulation of intermediates in the stroma or on the outside of the thylakoids. The fact that C1 is functionally equivalent to a stromal-targeting transit peptide shows that plastocyanin is imported into the chloroplast by way of the same mechanism as stromal proteins, and that import into and routing inside the chloroplasts are independent processes.

Fructan as a New Carbohydrate Sink in Transgenic Potato Plants.

Fructans are polyfructose molecules that function as nonstructural storage carbohydrates in several plant species that are important crops. We have been studying plants for their ability to synthesize and degrade fructans to determine if this ability is advantageous. We have also been analyzing the ability to synthesize fructan in relation to other nonstructural carbohydrate storage forms like starch. To study this, we induced fructan accumulation in normally non-fructan-storing plants and analyzed the metabolic and physiological properties of such plants. The normally non-fructan-storing potato plant was modified by introducing the microbial fructosyltransferase genes so that it could accumulate fructans. Constructs were created so that the fructosyltransferase genes of either Bacillus subtilis (sacB) or Streptococcus mutans (ftf) were fused to the vacuolar targeting sequence of the yeast carboxypeptidase Y (cpy) gene. These constructs were placed under the control of the constitutive cauliflower mosaic virus 35S promoter and introduced into potato tissue. The regenerated potato plants accumulated high molecular mass (>5 [times] 106 D) fructan molecules in which the degree of polymerization of fructose units exceeded 25,000. Fructan accumulation was detected in every plant tissue tested. The fructan content in the transgenic potato plants tested varied between 1 and 30% of dry weight in leaves and 1 and 7% of dry weight in microtubers. Total nonstructural neutral carbohydrate content in leaves of soil-grown plants increased dramatically from 7% in the wild type to 35% in transgenic plants. Our results demonstrated that potato plants can be manipulated to store a foreign carbohydrate by introducing bacterial fructosyltransferase genes. This modification affected photosynthate partitioning in microtubers and leaves and increased nonstructural carbohydrate content in leaves.

Reconstitution of mature plastocyanin from precursor apo-plastocyanin expressed in Escherichia coli.

The precursor plastocyanin from Silene pratensis (white campion) has been expressed in Escherichia coli. The precursor protein was accumulated in insoluble aggregates and partially purified as an apo-protein. The purified precursor apo-plastocyanin was processed to the mature apo-plastocyanin by chloroplast extracts. N-terminal amino-acid sequencing indicated that the processed protein was identical to the N-terminal amino-acid residues of mature plastocyanin that was deduced from the nucleotide sequence. The copper could be incorporated into the apo-plastocyanin of mature size in vitro, but could not into the precursor apo-plastocyanin under the same conditions. Absorption spectra and reduction potential of the reconstituted mature plastocyanin were indistinguishable from those of the purified spinach plastocyanin. The electron transfer activities of the reconstituted plastocyanin with both the Photosystem I reaction center (P700) and cytochrome f were almost the same as those of the purified spinach plastocyanin.

Genes and regulatory sequences of bacteriophage phi X174.

Fragments of the DNA of bacteriophage phi X174 were inserted in the plasmids pACYC177 and pBR322, in order to test the in vivo effects of separate phage genes and regulatory sequences. The phi X174 inserts were identified by recombination and complementation with phage mutants, followed by restriction enzyme analysis. The genes B, C, F and G can be maintained stably in the cell even when there is efficient expression of these viral genes. Recombinant plasmids with the complete genes D and E can only be maintained when the expression of these genes is completely blocked. Expression of complete H and J genes could not yet be demonstrated. The intact gene A was apparently lethal for the host cell, as it was never found in the recombinants. The genes F and G are expressed, even when they are not preceded by one of the well characterized viral or plasmid promoter sequences. Screening of the nucleotide sequence of phi X174 gives two promoter-like sequences just in front of the two genes. Viral sequences with replication signals (the phi X174 (+) origin of replication, the initiation site for complementary strand synthesis and the incompatibility sequence) appeared to be functional also when inserted in recombinant plasmids. A plasmid with the phi X (+) origin can be forced to a rolling circle mode of replication. The A protein produced by infecting phages works in trans on the cloned viral origin. The (-) origin can function as initiation signal for complementary strand synthesis during transduction of single-stranded plasmid DNA. The intracellular presence of the incompatibility sequence on a plasmid prevents propagation of infecting phages.

n' Protein activator sites of plasmid pBR322 are not essential for its DNA replication.

The lagging strand DNA synthesis of the Escherichia coli bacterial chromosome and plasmids is thought to be initiated by the mobile promotor, the primosome. This primosome is assembled at a specific site on single-stranded DNA. This process is initiated by the interaction of one of the at least seven components, the n' protein, with this site. Indeed n' protein activator sites are found in the plasmids Col E1 and pBR322. To investigate the in vivo function of these n' protein sites, deletion derivates of pBR322 were constructed in which the n' protein sites are removed. The deletion plasmids show no change in stability and only threefold reduction in copy number compared to pBR322. Using a transduction system for single-stranded plasmid DNA it was shown that no other specific initiation signals for lagging strand DNA synthesis were present in the deletion plasmids. It was concluded that the n' protein activator sites in pBR322 are not essential for its DNA replication in vivo.

Improved Performance of Transgenic Fructan-Accumulating Tobacco under Drought Stress.

Fructans are polyfructose molecules produced by approximately 15% of the flowering plant species. It is possible that, in addition to being a storage carbohydrate, fructans have other physiological roles. Owing to their solubility they may help plants survive periods of osmotic stress induced by drought or cold. To investigate the possible functional significance of fructans, use was made of transgenic tobacco (Nicotiana tabacum) plants that accumulate bacterial fructans and hence possess an extra sink for carbohydrate. Biomass production was analyzed during drought stress with the use of lines differing only in the presence of fructans. Fructan-producing tobacco plants performed significantly better under polyethylene-glycol-mediated drought stress than wild-type tobacco. The growth rate of the transgenic plants was significantly higher (+55%), as were fresh weight (+33%) and dry weight (+59%) yields. The difference in weight was observed in all organs and was particularly pronounced in roots. Under unstressed control conditions the presence of fructans had no significant effect on growth rate and yield. Under all conditions the total nonstructural carbohydrate content was higher in the transgenic plants. We conclude that the introduction of fructans in this non-fructan-producing species mediates enhanced resistance to drought stress.

Sucrose Represses the Developmentally Controlled Transient Activation of the Plastocyanin Gene in Arabidopsis thaliana Seedlings.

The plastocyanin (PC) gene of Arabidopsis thaliana is activated independently of light during early seedling development. In etiolated seedlings, PC mRNA levels increase transiently and a maximum dark level is reached after 2 d of growth in darkness. In etiolated transgenic seedlings carrying a chimeric PC-promoter: luciferase fusion gene, luciferase activity is similarly increased after 2 d of growth. The transient increase in PC mRNA and luciferase activity levels can be repressed by sucrose. Nonmetabolizable sugars and polyethylene glycol do not have a major effect on PC gene expression. Also, light-grown seedlings show a similar transient and sucrose-sensitive increase in PC mRNA levels and luciferase activity, as in dark-grown seedlings, but here expression levels are 15- fold higher. These findings suggest the presence of a sucrose-sensitive, developmentally controlled expression mechanism that operates independently of light.

Plastocyanin of Arabidopsis thaliana; isolation and characterization of the gene and chloroplast import of the precursor protein.

A genomic clone encoding the plastocyanin precursor was isolated from an Arabidopsis thaliana lambda EMBL3 library, with the help of a heterologous hybridization probe. The nucleotide (nt) sequence encoding the 171-amino acid precursor protein and 650 bp of the 5'-flanking region were determined. S1 nuclease mapping showed the Arabidopsis coding region to be uninterrupted and the transcript to possess an untranslated leader of approx. 30 nt. The 5' region of the gene contains a 25-bp direct repeat at a distance of 300 bp upstream from the ATG start codon. Southern analysis of several genomic digests shows the presence of a single copy of the plastocyanin gene in the Arabidopsis genome. In vitro synthesized pre-plastocyanin was used in import experiments with isolated pea chloroplasts. Plastocyanin was correctly directed to the thylakoid lumen and processed to the mature size. A clear single processing intermediate, as was found with the import of Silene pratensis pre-plastocyanin, seems to be absent.

Genomic integration system based on pBR322 sequences for the cyanobacterium Synechococcus sp. PCC7942: transfer of genes encoding plastocyanin and ferredoxin.

Synechococcus sp. PCC7942 recipient strains were constructed for the chromosomal integration of DNA fragments cloned in any pBR322-derived vector, which carries the ampicillin resistance (ApR) marker. The construction was based on the incorporation of specific recombination targets, the so-called 'integration platforms', into the chromosomal metF gene. These platforms consist of an incomplete bla gene (ApS) and the pBR322 ori separated from each other by a gene encoding an antibiotic (streptomycin or kanamycin) resistance (SmR or KmR). Recombination between a pBR322-derived donor plasmid and such a chromosomal platform results with high frequency in restoration of the bla gene and replacement of the chromosomal marker (SmR or KmR) by the insert of the donor plasmid. The integration into the platform depends on recombination between pBR322 ori and bla sequences only and is therefore independent of the DNA insert to be transferred. The desired recombinants are found by selection for a functional bla gene (ApR) and subsequent screening for absence of the chromosomal antibiotic marker. Gene transfer with this integration system was found to occur efficiently and reliably. Furthermore, the presence of the pBR322 ori in the platform allowed for 'plasmid rescue' of integrated sequences. The system was applied successfully for the transfer of the gene encoding plastocyanin (petE1) from Anabaena sp. PCC7937 and for the integration of an extra copy of the gene encoding ferredoxin I (petF1) from Synechococcus sp. PCC7942 itself.

Alginate regulatory and biosynthetic gene homologs in Pseudomonas putida WCS358: correlation with the siderophore regulatory gene pfrA.

A previous study [Venturi et al., Mol. Microbiol. 10 (1993) 63-73] demonstrated that the siderophore regulatory gene pfrA of Pseudomonas putida (Pp) WCS358 is highly similar and interchangeable with the alginate regulatory gene algQ (algR2) of P. aeruginosa (Pa). The algQ gene is physically linked to two other alginate regulators in the Pa chromosome, namely algR (algR1), a response regulator, and algP (algR3), a histone-like gene. In this study, we have identified the same genes and a similar genetic organization in the Pp chromosome. The two genes linked to pfrA, designated pprA and pprB, are similar to algR and algP, respectively. Chromosomal mutants of pprA and pprB were constructed showing that unlike pfrA, the two newly identified regulators are not involved in siderophore regulation. The pprA gene complemented a Pa algR mutant phenotype, suggesting that it could be involved in alginate gene regulation. The WCS358 strain is not producing alginate, but we demonstrated by Southern analysis that it also possesses, in addition to pprA and pprB, algD and algU (algT) gene homologs, two genes essential for alginate biosynthesis. Using an algD-xylE transcriptional fusion, we observed that the algD promoter is active in strain WCS358 and absolutely requires pfrA. The possibility that all five genes of Pp WCS358 are involved in alginate biosynthesis is discussed.

Kinetic analysis of translocation into isolated chloroplasts of the purified ferredoxin precursor.

Time courses of the import into isolated chloroplasts of the purified ferredoxin precursor were measured at different protein concentrations. Analysis of the initial import rates indicates the presence of one saturable import system with an apparent Km value of approximately 100 nM and a Vmax corresponding to the uptake of approximately 2.5 x 10(4) precursor proteins per minute per chloroplast. We conclude that the in vitro observed activity of the chloroplast protein import machinery, functioning independently from cytosolic factors, would be enough to allow chloroplast development at physiological rate.

Site-directed mutagenetic study on the role of negative patches on silene plastocyanin in the interactions with cytochrome f and photosystem I.

To investigate the role of two highly conserved negative patches, residues #42-45 and #59-61, on the surface of plant plastocyanin, six mutants were constructed by site-directed mutagenesis of the intermediate precursor gene from Silene pratensis. The mutants were designed systematically to incorporate positive charges into the negative patches, and the net charge on negative patches was modified from -4 to +1. Upon expression in Escherichia coli, the mutant proteins were correctly processed to the mature size and accumulated as holo-proteins. Absorption spectra, EPR, and redox potentials of the purified mutant proteins were almost indistinguishable from those of the wild-type. It was found that the electron transfer rate from cytochrome f to plastocyanin decreased exponentially as the net charge on the negative patch (#42-45) was increased, whereas the modification of the other negative patch (#59-61) had no effect. Ionic strength dependence studies indicated that the rate constants at infinite ionic strength did not change significantly among the wild-type and the six mutants, and the electrostatic attraction energies between plastocyanin and cytochrome f decreased when residues #42-45 were modified, whereas the modification of residues #59-61 had no effect. These results clearly indicated that only one (#42-45) of the two negative patches is involved in the transient complex formation with cytochrome f. Essentially similar results were observed for the electron transfer from plastocyanin to the photosystem I reaction center (P700), although in this case, slight participation of the negative patch (#59-61) is suggested.(ABSTRACT TRUNCATED AT 250 WORDS)

A siderophore peptide synthetase gene from plant-growth-promoting Pseudomonas putida WCS358.

Under iron limiting conditions, Pseudomonas putida WCS358 produces and secretes a fluorescent siderophore called pseudobactin 358 which consists of a nonapeptide linked to a fluorescent dihydroxy quinoline moiety. Previous studies have identified a major gene cluster involved in pseudobactin 358 biosynthesis and several regulators responsible for the activation of biosynthetic genes under iron starving conditions. In this study, we identified the promoter transcribing the pseudobactin 358 synthetase gene. Promoter deletion experiments have demonstrated that the DNA region downstream of the initiation of transcription site is necessary for proper promoter functioning. This promoter controls the expression of a gene designated ppsD which encodes a 2,247-residue protein, PpsD, which has a predicted molecular weight of 247,610 Da and contains two highly homologous domains of approximately 1000 amino acids each. ppsD::Tn5 mutants of strain WCS358 are unable to synthesise pseudobactin 358 and can be complemented when ppsD is provided in trans. It is concluded that ppsD is a peptide synthetase involved in the biosynthesis of the peptide moiety of pseudobactin 358. PpsD displays a very high degree of similarity (52% aa identity) with PvdD from P. aeruginosa, a non-ribosomal peptide synthetase involved in the biosynthesis of pyoverdine, the fluorescent siderophore produced by P. aeruginosa. It also displayed homology with other peptide synthetases from other micro-organisms involved in the biosynthesis of siderophores and peptide antibiotics.

Accumulation of fructose polymers in transgenic tobacco.

Fructan, a polyfructose molecule, is a storage compound in a limited number of plant species. Usually these species accumulate fructan with a low degree of polymerization (DP) and most of these plants have properties which preclude their use as a fructan source. With the eventual aim of allowing the accumulation of high DP fructans in non-fructan storing plants, we have investigated whether carbohydrate flow in the plant cell can be directed to produce this polymer. For this purpose the SacB gene from Bacillus subtilis, which encodes levansucrase, was modified and introduced into tobacco plants. Transgenic plants containing the sacB gene accumulate fructans. The size and properties of this fructan are similar to fructan produced by Bacillus subtilis, and is stable in plants. Although the level of fructan accumulation in the transgenic tobacco plants ranged from 3-8 percent of the dry weight, no levansucrase mRNA or protein could be detected in these plants. Extension of this work should permit the production of this high molecular weight biopolymer in crop plants for applications in food and non-food products.

Protein transport towards the thylakoid lumen: post-translational translocation in tandem.

Many proteins found in the chloroplast are synthesized in the cytoplasm as precursor molecules containing transit peptides. Proteins targeted to the stroma must pass through the two envelope membranes to reach their destination. Proteins located in the chloroplast lumen also have to be transferred across the thylakoid membrane. That is, lumen proteins must cross three biological membranes in order to reach their final location. Recent evidence shows that the routing of plastocyanin towards the lumen involves two post-translational transport processes mediated by two different regions of the transit peptide and two different processing proteases. It is postulated that the genetic information for the plastocyanin precursor, which already contained a signal peptide, was transferred from the endosymbiont to the nucleus. Then a chloroplast-specific targeting-peptide was added.

Identification of potential regulatory elements in the far-upstream region of the Arabidopsis thaliana plastocyanin promoter.

The far-upstream region of the Arabidopsis thaliana plastocyanin (Pc) promoter acts positively on transcription. This -1580 to -710 region (relative to the translation start site) has enhancer-like properties since it is also functional when situated downstream of the gene. Using tobacco nuclear extracts, this region was tested for protein-binding sites. Two short binding sequences were identified. The AT-rich sequence separating these binding sites shows extensive homology to the sequences separating the paired GT-1-binding sites of the pea rbcS-3A promoter. The requirements for complex formation strongly suggest that a GT-1-like protein binds to the two identified boxes in the Pc promoter. Sequence comparisons revealed that both boxes fit within the moderate consensus sequence needed for GT-1-binding. This GT-1-like DNA-binding activity is present in light-grown as well as in dark-adapted plants. Therefore, the possible role for GT-1 in light regulation of transcription does not depend upon its de novo synthesis. In some of the gel mobility shift assays, an additional DNA-protein complex was formed. The formation of this complex was only observed if the heteropolymer poly(dAdT).poly(dAdT) was used as a non-specific competitor and was dependent on the CpG density of the probe used.