The Arabidopsis chloroplast ribosomal protein L21 is encoded by a nuclear gene of mitochondrial origin.

Many chloroplast genes of cyanobacterial origin have been transferred to the nucleus during evolution and their products are re-addressed to chloroplasts. The RPL21 gene encoding the plastid r-protein L21 has been lost in higher plant chloroplast genomes after the divergence from bryophytes. Based on phylogenetic analysis and intron conservation, we now provide evidence that in Arabidopsis a nuclear RPL21c gene of mitochondrial origin has replaced the chloroplast gene. The control of expression of this gene has been adapted to the needs of chloroplast development by the acquisition of plastid-specific regulatory promoter cis-elements.

The plastid chromosome of spinach (Spinacia oleracea): complete nucleotide sequence and gene organization.

The chloroplast chromosome of spinach (Spinacia oleracea) is a double-stranded circular DNA molecule of 150,725 nucleotide pairs. A comparison of this chromosome with those of the three other autotrophic dicotyledons for which complete DNA sequences of plastid chromosomes are available confirms a conserved overall structure. Three classes of open reading frames were distinguished: (1) genes of known function which include 108 unique loci, (2) three hypothetical chloroplast reading frames (ycfs) that are highly conserved interspecifically, and (3) species-specific or rapidly diverging 'open reading frames'. A detailed transcript study of one of the latter (ycf15) shows that these loci may be transcribed, but do not constitute protein-coding genes.

Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana.

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.

A second, substrate-dependent site of protein import into chloroplasts.

Chloroplasts must import a large number of proteins from the cytosol. It generally is assumed that this import proceeds for all stromal and thylakoid proteins in an identical manner and is caused by the operation of two distinctive protein import machineries in the outer and inner plastid envelope, which form the general import site. Here we show that there is a second site of protein translocation into chloroplasts of barley, tobacco, Arabidopsis thaliana, and five other tested monocotyledonous and dicotyledonous plant species. This import site is specific for the cytosolic precursor of the NADPH:protochlorophyllide (Pchlide) oxidoreductase A, pPORA. It couples Pchlide synthesis to pPORA import and thereby reduces the actual level of free Pchlide, which, because of its photodynamic properties, would be destructive to the plastids. Consequently, photoprotection is conferred onto the plant.

Progress in Arabidopsis genome sequencing and functional genomics.

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.

Characterization of a ubiquitous expressed gene family encoding polygalacturonase in Arabidopsis thaliana.

Pectin, as one of the major components of plant cell wall, has been implicated in many developmental processes occurring during plant growth. Among the different enzymes known to participate in the pectin structure modifications, polygalacturonase (PG) activity has been shown to be associated with fruit ripening, organ abscission and pollen grain development. Until now, sequence analyses of the deduced polypeptides of the plant PG genes allowed their grouping into three clades corresponding to genes involved in one of these three activities. In this study, we report the sequence of three genomic clones encoding PG in Arabidopsis thaliana. These genes, together with 16 other genes present in the databases form a large gene family, ubiquitously expressed, present on the five chromosomes with at least two gene clusters on chromosomes II and V, respectively. Phylogenetic analyses suggest that the A. thaliana gene family contains five classes of genes, with three of them corresponding to the previously defined clades. Comparison of positions and numbers of introns among the A. thaliana genes reveals structural conservation between genes belonging to the same class. The pattern of intron losses that could have given rise to the PG gene family is consistent with a mechanism of intron loss by replacement of an ancestral intron-containing gene with a reverse-transcribed DNA copy of a spliced mRNA. Following this event of intron loss, the acquisition of introns in novel positions is consistent with a mechanism of intron gain at proto-splice sites.

Differential expression of a polygalacturonase gene family in Arabidopsis thaliana.

By systematic sequencing of a flower bud cDNA library from Arabidopsis thaliana, we have identified four cDNAs encoding polygalacturonase. The corresponding genes, together with seven other A. thaliana genes present in the databases, form a small gene family. Sequence comparisons of the deduced polypeptides within the gene family or with other plant polygalacturonases allow classification of the genes into different clades. Five polygalacturonases, including all those isolated from the flower buds, are closely related to the enzyme in pollen. Of the six remaining polygalacturonases, three are more closely related to the abscission-specific type of enzyme and two others to the fruit polygalacturonase. The last one is more distantly related to the others and might correspond to a new type of polygalacturonase. Expression of the different genes was analysed on Northern blots and by a PCR-based strategy. Results indicate that if, as expected, the cDNAs isolated from the flower bud library are strongly expressed in pollen, other genes are expressed at a low level in young developing tissues, such as in seedlings and roots, suggesting that they could be implicated in the cell wall modifications observed during cell elongation and/or expansion which occur in these tissues.

Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast terminal oxidase associated with phytoene desaturation.

The immutans (im) mutant of Arabidopsis shows a variegated phenotype comprising albino and green somatic sectors. We have cloned the IM gene by transposon tagging and show that even stable null alleles give rise to a variegated phenotype. The gene product has amino acid similarity to the mitochondrial alternative oxidase. We show that the IM protein is synthesized as a precursor polypeptide that is imported into chloroplasts and inserted into the thylakoid membrane. The albino sectors of im plants contain reduced levels of carotenoids and increased levels of the carotenoid precursor phytoene. The data presented here are consistent with a role for the IM protein as a cofactor for carotenoid desaturation. The suggested terminal oxidase function of IM appears to be essential to prevent photooxidative damage during early steps of chloroplast formation. We propose a model in which IM function is linked to phytoene desaturation and, possibly, to the respiratory activity of the chloroplast.

Sequence and analysis of chromosome 4 of the plant Arabidopsis thaliana.

The higher plant Arabidopsis thaliana (Arabidopsis) is an important model for identifying plant genes and determining their function. To assist biological investigations and to define chromosome structure, a coordinated effort to sequence the Arabidopsis genome was initiated in late 1996. Here we report one of the first milestones of this project, the sequence of chromosome 4. Analysis of 17.38 megabases of unique sequence, representing about 17% of the genome, reveals 3,744 protein coding genes, 81 transfer RNAs and numerous repeat elements. Heterochromatic regions surrounding the putative centromere, which has not yet been completely sequenced, are characterized by an increased frequency of a variety of repeats, new repeats, reduced recombination, lowered gene density and lowered gene expression. Roughly 60% of the predicted protein-coding genes have been functionally characterized on the basis of their homology to known genes. Many genes encode predicted proteins that are homologous to human and Caenorhabditis elegans proteins.

COP1b, an isoform of COP1 generated by alternative splicing, has a negative effect on COP1 function in regulating light-dependent seedling development in Arabidopsis.

COP1 is a negative regulator of Arabidopsis light-dependent development. Mutation of the COP1 locus causes constitutive photomorphogenesis in the dark. Here, we report the identification of an isoform of the COP1 protein, named COP1b, which is generated by alternative splicing. COP1b has a 60-amino acid deletion in the WD-40 repeat domain relative to the full-length COP1. This splicing step is light-independent and takes place mostly in mature seeds and in germinating seedlings. Transgenic Arabidopsis plants that overexpress COP1b show a de-etiolated phenotype in the dark, with a short hypocotyl, open and developed cotyledons. The transgenic seedlings are adult-lethal. These phenotypes closely resemble that of severe cop-1 mutants, indicating that COP1b has a dominant negative effect on COP1 function.

S2F, a leaf-specific trans-acting factor, binds to a novel cis-acting element and differentially activates the RPL21 gene.

Tissue-specific factors control the differential expression of nuclear genes encoding plastid proteins. To identify some of these factors, the light-independent spinach RPL21 gene encoding the plastid ribosomal protein L21 was chosen as a model. The RPL21 promoter organization was defined by transient and stable transfections of RPL21 promoter deletion mutants fused to a reporter gene. The following results were obtained. (1) We identified a strong core promoter, spanning the transcription start site region, sufficient to drive high levels of gene expression. (2) We identified two non-overlapping positive and negative domains, located upstream from the core promoter region, that modulate core promoter activity independently of light. (3) We found that the positive domain contains a new cis-acting element, the S2 site, related to but different from the light-responsive GT-1 binding site. We show that the S2 site binds a leaf-specific nuclear factor (named S2F). The S2 site is conserved in the promoter region of many nuclear genes encoding plastid proteins. Experiments with transgenic tobacco plants confirmed that the S2 site is critical for positive domain activity in leaf tissues. The S2 site is thus identified as a new tissue-specific, light-independent regulatory element.

The mechanism of GT element-mediated cell type-specific transcriptional control.

Promoter studies have revealed that sequences related to the GT-1 binding site, known as GT elements, are conserved in plant nuclear genes of diverse functions. In this work, we addressed the issue of whether GT elements are involved in cell type-specific transcriptional regulation. We found that the inactivation of GT-1 site-mediated transcription in roots is correlated with the absence of the GT-1 binding activity in root extracts. In addition, the mutation of the related GT-1 (from the pea rbcs-3A) and the S1F (from the spinach rps1) sites resulted in an increase of their transcriptional activity in roots that contain a distinct GT element-binding factor, referred to as RGTF. Although specific to GT elements, RGTF has a different sequence requirement and a lower sequence specificity than GT-1. Interestingly, RGTF has a higher binding affinity to the mutant GT-1 and S1F sites than to the wild-type sequences. This correlation suggests that RGTF may have some role in transcriptional regulation in roots. Furthermore, root cellular protein extracts contain an inhibitory activity that prevents GT-1 from binding to DNA. This helps to explain the absence of the GT-1 binding activity in roots in which the gene of GT-1 is expressed. Together, these data suggest that the cell type-specific transcription modulation by GT elements is achieved by using two different strategies.

Sequence analysis of an 81 kb contig from Arabidopsis thaliana chromosome III.

The nucleotide sequence of a 81 493 bp contig from Arabidopsis thaliana chromosome III has been determined together with 11 corresponding cognate cDNAs. Analysis of the finished sequence and comparison with public databases indicates a gene density of one gene per 4527 bp and identifies 17 novel genes, 10 of which are totally unknown or have no well-defined function. In addition, the contig contains part of a non-LTR retrotransposon and large direct and inverted repeats. Contig analysis also provides information on the structure and genomic organization of plant genes.

Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs.

Nearly 7000 Arabidopsis thaliana-expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.

Cis-acting elements and expression pattern of the spinach rps22 gene coding for a plastid-specific ribosomal protein.

In order to study the regulation of nuclear genes coding for plastid ribosomal proteins, we have analysed the promoter region of spinach rps22 using both in vitro and in vivo approaches. By footprinting analyses, we have identified eight DNA elements interacting with spinach leaf nuclear factors in the 300 bp promoter region upstream of the transcription start site. Among these elements, four are short AT-rich sequences and one is identical to the Hex motif characterized initially in wheat histone genes. In transgenic tobacco plants, the reporter gene coding for the beta-glucuronidase (GUS) directed by a 1.2 kb upstream region of rps22 was expressed in several plant organs, with high levels in leaf mesophyll, embryo cotyledons and root meristematic cells and very low levels in other cell types. Interestingly, when deleted to -295, the promoter, which contained all the foot-printed elements, was still able to confer the same expression pattern, although the activity was relatively lower than with the 1.2 kb promoter. When deleted further to -154, the promoter, from which the AT-rich elements were eliminated, loses its activity almost completely, suggesting that these AT-rich elements are important for the rps22 promoter activity. Altogether, our results show that rps22 gene expression is controlled by specific cis elements not present in other nuclear-encoded plastid ribosomal protein genes studied so far.

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.

Molecular cloning of a small DNA binding protein with specificity for a tissue-specific negative element within the rps1 promoter.

A cDNA encoding a specific binding activity for the tissue-specific negative cis-element S1F binding site of spinach rps1 was isolated from a spinach cDNA expression library. This cDNA of 0.7 kb encodes an unusual small peptide of only 70 amino acids, with a basic domain which contains a nuclear localization signal and a putative DNA binding helix. This protein, named S1Fa, is highly conserved between dicotyledonous and monocotyledonous plants and may represent a novel class of DNA binding proteins. The corresponding mRNA is accumulated more in roots and in etiolated seedlings than in green leaves. This expression pattern is correlated with the tissue-specific function of the S1F binding site which represses the rps1 promoter preferentially in roots and in etiolated plants.

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.

S1F binding site is related to but different from the light-responsive GT-1 binding site and differentially represses the spinach rps1 promoter in transgenic tobacco.

Nuclear genes encoding plastid ribosomal proteins are more highly expressed in leaves than in roots. This leaf-specific induction seems to be light-independent. We have previously characterized a spinach nuclear factor S1F binding to a cis-element within the rps1 promoter, which negatively regulates both the rps1 and the cauliflower mosaic virus 35S promoters in transient expression assays. Here, we show that the S1F binding site is related to but different from the light-responsive Box II of the pea rbcS-3A promoter, which is recognized by the nuclear factor GT-1. Transgenic plant analyses showed that the S1F site tissue-specifically represses the rps1 promoter in roots as well as in etiolated seedlings. We suggest that the GT-1-related S1F binding site is responsible, at least in part, for the transcriptional repression of rps1 in nonphotosynthetic tissues such as roots.

The ubiquitous presence of exopolygalacturonase in maize suggests a fundamental cellular function for this enzyme.

Exopolygalacturonase (exoPG) is a pectin-degrading enzyme abundant in maize pollen. Using immunochemistry and in situ hybridization it is shown that in addition to its presence in pollen, exoPG is also present in sporophytic tissues, such as the tapetum and mesophyll cells. The enzyme is located in the cytoplasm of pollen and of some mesophyll cells. In other mesophyll cells, the tapetum and the pollen tube, exoPG is located in the cell wall. The measurement of enzyme activity shows that exoPG is ubiquitous in the vegetative organs. These results suggest a general function for exoPG in cell wall edification or degradation. ExoPG is encoded by a closely related multigene family. The regulation of the expression of one of the exoPG genes was analyzed in transgenic tobacco. Reporter GUS activity was detected in anthers, seeds and stems but not in leaves or roots of transgenic plants. This strongly suggests that the ubiquitous presence of exoPG in maize is the result of the expression of different exoPG genes.