Characterization of the plastid-specific germination and seedling establishment transcriptional programme.

Upon imbibition, dry seeds rapidly gain metabolic activity and the switching on of a germination-specific transcriptional programme in the nucleus goes ahead, with the induction of many nucleus-encoded transcripts coding for plastid-localized proteins. Dedifferentiated plastids present in dry seeds differentiate into chloroplasts in cotyledons and into amyloplasts in the root and in the hypocotyl, raising the question of whether the beginning of a new plant's life cycle is also characterized by specific changes in the plastid transcriptional programme. Here the plastid transcriptome is characterized during imbibition/stratification, germination, and early seedling outgrowth. It is shown that each of these three developmental steps is characterized by specific changes in the transcriptome profile, due to differential activities of the three plastid RNA polymerases and showing the integration of plastids into a germination-specific transcriptional programme. All three RNA polymerases are active during imbibition; that is, at 4 °C in darkness. However, activity of plastid-encoded RNA polymerase (PEP) is restricted to the rrn operon. After cold release, PEP changes specificity by also transcribing photosynthesis-related genes. The period of germination and radicle outgrowth is further characterized by remarkable antisense RNA production that diminishes during greening when photosynthesis-related mRNAs accumulate to their highest but to very different steady-state levels. During stratification and germination mRNA accumulation is not paralleled by protein accumulation, indicating that plastid transcription is more important for efficient germination than translation.

Cell cycle-dependent modulation of FtsZ expression in synchronized tobacco BY2 cells.

In higher plants, the FtsZ protein, the ancestor of tubulin, has been shown to be implicated in both proplastid division, which occurs in dividing cells and in the division of the differentiated plastids present in non-dividing cells. Here we report studies on the expression of the two FtsZ gene families in higher plants, FtsZ1 and FtsZ2, in non-synchronized and synchronized tobacco BY2 cells. We have isolated and characterized members of each gene family from Nicotiana tabacum. Specific cDNA probes for each tobacco FtsZ gene family and polyclonal antibodies specific for the FtsZ1 and FtsZ2 proteins were obtained in order to determine mRNA and protein levels. A constant level of FtsZ1 and FtsZ2 transcripts and proteins was observed in non-synchronized cell cultures. However, a complex pattern of expression of both gene families was observed during the cell cycle in synchronized cells, with mRNA and protein levels peaking during cell division, thus implying that the FtsZ proteins may be involved in plastid transmission to the two daughter cells.

Regulation of rDNA transcription in plastids of higher plants.

Plastid rDNA expression changes with plastid differentiation, plant development and plant growth conditions. Plastid rDNA operons are preceded by different promoter structures that are recognised by different types of RNA polymerase in a species-specific and development-dependent manner. Present knowledge on regulation of rDNA transcription obtained with plant material corresponding to different developmental stages and/or different plant species is summarised. Results indicate the creation of new and unique transcriptional regulatory mechanisms during the evolutionary integration process of the cyanobacterial ancestor into the present-day multi-cellular organism.

Extra-ribosomal function(s) of the plastid ribosomal protein L4 in the expression of ribosomal components in spinach.

We have previously characterised the cDNA corresponding to the nucleus-encoded, plastid ribosomal protein L4 from spinach. The L4 protein belongs to the group of ribosomal proteins for which extra-ribosomal functions have been demonstrated in prokaryotes. In general, these functions are concerned with the expression of ribosomal components. In order to analyse whether the plastid L4 protein might also have (an) extra-ribosomal function(s) we have produced the plastid L4 protein as a thioredoxin fusion protein and analysed its role in both prokaryotic (E. coli) and plastid systems. We found that the plastid L4 protein can replace the E. coli L4 protein in the NusA-dependent attenuation control of the E. coli S10 operon by stabilising stalled transcription complexes in a NusA-dependent reaction. In plastids, the L4 protein inhibits transcription of the rrn operon. Our results thus suggest extra-ribosomal function(s) for the plastid L4 protein in the expression of ribosomal components.

Regulation of plastid rDNA transcription by interaction of CDF2 with two different RNA polymerases.

The plastid genome is known to be transcribed by a plastid-encoded prokaryotic-type RNA polymerase (PEP) and by a nucleus-encoded phage-type RNA polymerase (NEP). The spinach plastid rrn operon promoter region harbours three different, overlapping promoters. Two of them are of the prokaryotic type. The third promoter is a non-consensus-type NEP promoter. We separated three different transcriptional activities from spinach chloroplasts: PEP, the phage-type RNA polymerase NEP-1, and a third, hitherto undescribed transcriptional activity (NEP-2). NEP-2 specifically transcribes the rrn operon in the presence of the transcription factor CDF2. CDF2 was previously shown to recruit PEP to the rrn promoter to repress transcription. Together, our results suggest the existence of a third RNA polymerase in plastids and a mechanism of rDNA transcriptional regulation that is based on the interaction of the transcription factor CDF2 with two different transcriptional systems.

Evolutionary conservation of C-terminal domains of primary sigma(70)-type transcription factors between plants and bacteria.

Three different cDNAs coding for putative plant plastid sigma(70)-type transcription initiation factors have recently been cloned and sequenced from Arabidopsis thaliana. We have analyzed the evolutionary conservation of function(s) of the N-terminal and C-terminal halves of these three sigma factors by in vitro transcription studies using heterologous transcription systems and by complementation assays using Escherichia coli thermosensitive rpoD mutants. Our results indicate differences and similarities of the three plant factors and their prokaryotic ancestors. The functions of the N-terminal parts of the plant sigma factors are considerably different from the function of the N-terminal part of the principal sigma(70) factor of E. coli. On the other hand, the C-terminal parts have kept at least two characteristics when compared with their prokaryotic ancestors: 1) they can distinguish between different promoter structures, and 2) one of them is capable of fully complementing E. coli rpoD mutants, i.e. recognizing all essential E. coli promoters that are used by the E. coli principal sigma(70) factor. This shows for the first time in vivo a strong evolutionary conservation of cis- and trans-acting elements between the prokaryotic and the plant plastid transcriptional machinery.

Functional analysis of two maize cDNAs encoding T7-like RNA polymerases.

We have characterized two maize cDNAs, rpoTm and rpoTp, that encode putative T7-like RNA polymerases. In vivo cellular localization experiments using transient expression of the green fluorescent protein suggest that their encoded proteins are targeted exclusively to mitochondria and plastids, respectively. An antibody raised against the C terminus of the rpoTp gene product identified mitochondrial polypeptides of approximately 100 kD. Their presence was correlated with RNA polymerase activity, and the antibody inhibited mitochondrial in vitro transcription activity. Together, these results strongly suggest that the product of rpoTm is involved in maize mitochondrial transcription. By contrast, immunoblot analysis and an antibody-linked polymerase assay indicated that rpoTp specifies a plastid RNA polymerase component. A quantitative reverse transcription-polymerase chain reaction assay was used to study the transcription of rpoTp and rpoTm in different tissues and under different environmental conditions. Although both genes were constitutively expressed, rpoTm transcripts were generally more prevalent in nonphotosynthetic tissues, whereas an increase in rpoTp transcripts paralleled chloroplast development. We suggest that these two genes encode constitutive components of the organelle transcription machinery but that their expression is nonetheless subject to modulation during plant development.

The nuclear RPL4 gene encodes a chloroplast protein that co-purifies with the T7-like transcription complex as well as plastid ribosomes.

We have cloned and sequenced the cDNA and the gene coding for plastid ribosomal protein L4 (RPL4) from two higher plant species, spinach and Arabidopsis thaliana. Ribosomal protein L4 is one of the ribosomal proteins for which extraribosomal functions in transcriptional regulation has been demonstrated in prokaryotes. Sequence comparison of the two plant cDNAs and genes shows that the RPL4 gene has acquired a remarkable 3' extension during evolutionary transfer to the nuclear genome. This extension harbors an intron and codes for a glutamic and aspartic acid-rich amino acid sequence that resembles highly acidic C-terminal tails of some transcription factors. Co-purification of ribosomal protein L4 with plastid RNA polymerase and transcription factor CDF2 using different purification protocols as well as the surprising amino acid sequence of the L4 protein make it a likely candidate to play a role in plastid transcriptional regulation.

Clp protease complexes and their diversity in chloroplasts.

The Clp proteases represent a large, ancient ATP-dependent protease family which in higher plants is known to be located in chloroplasts. The soluble, presumably multisubunit, enzyme of the organelle stroma is of dual genetic origin. It consists of a nuclear-encoded, regulatory subunit ClpC, which is an ATPase, and a plastid-encoded proteolytic subunit ClpP, which is a serine protease. An additional, nuclear-encoded proteolytic subunit resembling ClpP has been recently reported from tomato (Schaller and Ryan, 1995 plant gene Register 95-00). We demonstrate that in both tomato Lycopersicon esculentum Mill. and Arabidopsis thaliana, (L.) Heynh. the nuclear-encoded ClpP (nClpP) is made as a precursor molecule that can be imported into isolated intact chloroplasts of spinach (Spinacia oleracea L.) and processed in two or three steps, respectively, to the size of the authentic protein. Furthermore, both gel electrophoresis under non-denaturing conditions and size-exclusion chromatography verified that the three proteins can form distinct heteromeric supramolecular complexes of approximately 860, 1380 and 1700 kDa (probably also of 600 kDa) molecular mass. The size ranges of the former two are reminiscent of those of Clp complexes described from Escherichia coli. In addition, various complexes between 160 and 560 kDa are detectable with the individual components. Both the processing "intermediates" and the mature nClpP are found in assembled form.

Organ-specific transcription of the rrn operon in spinach plastids.

The spinach rrn operon is used as a model system to study transcriptional regulation in higher plant photosynthetic and non-photosynthetic plastids. We performed capping experiments to determine whether P1, PC, or P2 promoters are employed for rrn transcription start sites in cotyledon and root tissues. By using a new method of analysis of capped RNA we demonstrate for the first time that 1) in both organs the rrn operon is expressed in a constitutive manner by cotranscription with the preceding tRNA(GAC)Val gene, and 2) the PC transcription start site is used only in cotyledons and leaves, i.e. we demonstrate the organ-specific usage of a plastid promoter. Both start sites, PC and that of the tRNA(GAC)Val cotranscript, lack Escherichia coli-like consensus sequences. The cotranscript is initiated 457 base pairs upstream of the tRNA(GAC)Val gene. The PC-specific DNA-binding factor, CDF2, is not detectable in root tissues confirming its regulatory role in PC-initiated rrn expression and the organ specificity of PC expression. Furthermore, our results show that rrn operon expression patterns differ in spinach and tobacco indicating species-specific transcriptional regulation of plant plastid gene expression.

The expression of nuclear genes encoding plastid ribosomal proteins precedes the expression of chloroplast genes during early phases of chloroplast development.

The development of different plant organs (root, hypocotyl, and cotyledons) during seed germination is connected with the transformation of proplastids, which are found in embryonic and meristematic tissues, into amyloplasts in root tissues and into chloroplasts in cotyledons. We have analyzed the expression of nuclear and plastid genes coding for the plastid translational apparatus during the first 7 d of Spinacia oleracea development. Results show that the nuclear genes (rps1, rps22, rpI21, and rpI40) are expressed from the 1st d of seed imbibition and precede transcription of the chloroplast-encoded genes (photosynthetic and nonphotosynthetic), which starts the 3rd d after the beginning of imbibition. Transcription from the leaf-/cotyledon-specific P1 promoter of the rpI21 gene starts on the first imbibition day. Inhibition of chloroplast biogenesis by bleaching in the presence of norflurazon has no influence on the expression from this P1 promoter, suggesting that the onset of transcription of nuclear gene rpI21 is independent of a plastid signal.

Regulation of rDNA transcription in chloroplasts: promoter exclusion by constitutive repression.

Spinach chloroplasts contain two types of RNA polymerases. One is multimeric and Escherichia coli-like. The other one is not E. coli-like and might represent a monomeric enzyme of 110 kD. The quantitative relation of the two polymerases changes during plant development. This raises the question, how are plastid genes transcribed that contain E. coli-like and non-E. coli-like promoter elements during developmental phases when both enzymes are present? Transcription of the spinach plastid rrn operon promoter is initiated at three sites: P1, PC, and P2. P1 and P2 are preceded by E. coli-like promoter elements that are recognized by E. coli RNA polymerase in vitro. However, in vivo, transcription starts exclusively at PC. We analyzed different promoter constructions using in vitro transcription and gel mobility-shift studies to understand why P1 and P2 are not used in vivo. Our results suggest that the sequence-specific DNA-binding factor CDF2 functions as a repressor for transcription initiation of the E. coli-like enzyme at P1 and P2. We propose a mechanism of constitutive repression to keep the rrn operon in all developmental phases under the transcriptional control of the non-E. coli-like RNA polymerase.

The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single-subunit enzyme or catalytic core of multimeric enzyme complexes?

Highly purified RNA polymerase preparations from spinach chloroplasts contain seven major polypeptides of 150, 145, 110, 102, 80, 75, and 38 kDa. I find that RNA polymerase activity can be separated under defined conditions into three different fractions by heparin-Sepharose chromatography. Immunological analysis has shown that the first fraction contains RNA polymerase activity associated with all seven major polypeptides, and other studies have shown that some of these polypeptides (150, 145, 80, and 38 kDa) are associated with an RNA polymerase similar to the Escherichia coli enzyme. However, similar analyses of the remaining fractions show activity associated only with the 110-kDa polypeptide, suggesting the existence of a second kind of chloroplast RNA polymerase. Samples of this 110-kDa polypeptide purified by SDS/PAGE actively synthesize RNA in a reaction dependent on a supercoiled DNA template and the four ribonucleoside triphosphates. Hence, this polypeptide has all of the properties expected of a single-subunit RNA polymerase of the T7 bacteriophage type.

Structure and expression of the nuclear gene coding for the chloroplast ribosomal protein L21: developmental regulation of a housekeeping gene by alternative promoters.

We have cloned and sequenced the nuclear gene of the chloroplast ribosomal protein L21 (rpl21) of Spinacia oleracea. The gene consists of five exons and four introns. All introns are located in the sequence which corresponds to the Escherichia coli-like central core of the protein. L21 mRNA is present in photosynthetic (leaves) and nonphotosynthetic (roots and seeds) plant organs, although large quantitative differences exist. Primer extension and S1 nuclease mapping experiments revealed the existence of two types of transcripts in leaves. The two corresponding start sites were defined as P1 and P2. In roots and seeds, we found only the shorter of the two transcripts (initiated at P2). The nucleotide sequence surrounding P2 resembles promoters for housekeeping and vertebrate r-protein genes. Analysis of several promoter constructions by transient expression confirmed that both transcripts originate from transcription initiation. Results are interpreted to mean that the expression of the rpl21 gene is regulated by alternative promoters. One of the promoters (P2) is constitutive, and the other one (P1) is specifically induced in leaves, i.e., its activation should be related to the transformation of amyloplasts or proplastids to chloroplasts. The gene thus represents the first example of a housekeeping gene which is regulated by the organ-specific usage of alternative promoters. Primer extension analysis and S1 nuclease mapping of another nucleus-encoded chloroplast ribosomal protein gene (rps1) give evidence that the same type of regulation by two-promoter usage might be a more general phenomenon of plant chloroplast-related ribosomal protein genes. Preliminary results indicate that presence of conserved sequences within the rpl21 and rps1 promoter regions which compete for the same DNA binding activities.

Characterization of a protein binding sequence in the promoter region of the 16S rRNA gene of the spinach chloroplast genome.

By means of mobility-shift assays and Exonuclease III mapping we have determined a 14 bp sequence (named CDF2 binding site) located in front of the 16S rRNA initiation start site which is protected by a spinach chloroplast extract. This region does not include neither one of the two '-35' nor of the two '-10' E. coli-like promoter elements which are recognised by E. coli RNA polymerase in vitro. The CDF2 binding site is specifically recognized by two small polypeptides which migrate corresponding to 35 and 33 kDa respectively as shown by UV cross-linking experiments. In vivo transcription initiation of the 16S rRNA gene occurs 13 nucleotides downstream of the 14 bp sequence and is different from the transcription start site which is used by E.coli polymerase in vitro.

Quantification of DNA-dependent RNA polymerase subunits and initiation factor(s) by antibody-linked polymerase assays.

The antibody-linked polymerase assay is a method which allows one to assign RNA polymerase activity to SDS-denatured polypeptides on nitrocellulose membranes using antibodies which were raised against only partially purified polymerase preparations. Here we show that with this method not only enzyme subunits but also initiation factor(s) can be determined in crude homogenates. Moreover the determination is quantitative. Therefore changes in the amount of individual polymerase subunits and factor(s) can be visualized within different crude homogenates.