INTRODUCTION . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475 CHLOROPLAST DEVELOPMENT AND PLASTID DIVERSITY 477 Proplastids . . . . . . . . . .... . . ... 477 Formation of Chloroplasts from Proplastids . . . . . . . . . . ...... . . . . . . . . . ... . . . . . . . . . 477 Generation of Plastid Diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. 478 PLASTID CODING CAPACITY AND GENOME ORGANIZATION 479 PHASES OF CHLOROPLAST DEVELOPMENT . . " . . . . " . . . . . . . . . . . . . . ...... "",,. . . . . . . . . 48 1 Formation of the Shoot and Initiation of Leaf Meristems . . . ... . . . . . . . . . . .. . . . . . . . . . . ... . 481 Leaf Mesophyll Cell Development . . . ..... . . . . . . . . . . " . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " . . . . . . . . . 482 REGULATION OF CHLOROPLAST PROTEIN COMPLEX BIOSYNTHESIS......... 483 PLASTID TRANSCRIPTION AND THE DETERMINATION OF RNA LEVELS..... 485 Regulation of Chloroplast Transcription . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . 485 Posttranscriptional Mechanisms that Alter RNA Levels 486 REGULATION OF CHLOROPLAST TRANSLATION ... ..... 488 The Cytoplasmic Control Principle. . . . . . .. . . .. . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 490 Regulation of Chlorophyll-Apoprotein Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 490 CONCLUDING REMARKS . . . . . . ... . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 493

Chloroplast Development and Gene Expression

The nuclear genome of the model plant Arabidopsis thaliana contains a small gene family consisting of three genes encoding RNA polymerases of the single-subunit bacteriophage type. There is evidence that similar gene families also exist in other plants. Two of these RNA polymerases are putative mitochondrial enzymes, whereas the third one may represent the nuclear-encoded RNA polymerase (NEP) active in plastids. In addition, plastid genes are transcribed from another, entirely different multisubunit eubacterial-type RNA polymerase, the core subunits of which are encoded by plastid genes [plastid-encoded RNA polymerase (PEP)]. This core enzyme is complemented by one of several nuclear-encoded sigma-like factors. The development of photosynthetically active chloroplasts requires both PEP and NEP. Most NEP promoters show certain similarities to mitochondrial promoters in that they include the sequence motif 5'-YRTA-3' near the transcription initiation site. PEP promoters are similar to bacterial promoters of the -10/-35 sigma 70 type.

Organellar RNA polymerases of higher plants.

Chloroplast gene expression: How plants turn their plastids on

Wilhelm Gruissem Department of Botany University of California Berkeley, California 94720 Plant and animal cells are fundamentally very similar. Be- sides the organelles found in both cell types, however, plant cells contain a unique class of organelles, the plastids. Since the early discovery by Correns (1909) and Baur (1909) that mutations affecting plastid phenotypes in higher plants frequently exhibit non-Mendelian inheri- tance, research on the DNA of this organelle has now yielded the complete sequence of the plastid genomes from tobacco (Shinozaki et al., 1986) and liverwort (Oh- yama et al., 1986). Plastids exist in a number of different forms with different functions, but the green chloroplast was the first to be discovered, and is the best studied of all plastids. The diversity of plastid types is controlled by the developmental program of the plant, which indicates that there must be a significant flow of information be- tween two separate genetic compartments in the cell. The use of chloroplasts to study photosynthesis and the in- tricacy of photosynthetic complexes has yielded new infor- mation on controls of organelle gene expression and the communication of different genomes in eukaryotic cells. In developing plants, chloroplasts are derived from small proplastids, which are the undifferentiated plastids present in meristematic cells. During the development of chloroplasts in photosynthetic tissues, photosynthetic electron-transfer components are assembled into pho- tosystems I and II, cytochrome bsf, and ATP synthase complexes, each of which consists of up to 20 polypep- tides. Proplastids and chloroplasts can also differentiate into specialized plastid types that assume other functions in nonphotosynthetic plant organs of higher plants, such as amyloplasts in roots and tubers or chromoplasts in many flowers and fruits. Photosynthesis, together with other plastid functions, requires the products of several hundred genes, of which only about 120 are present in the approximately 150 kb chloroplast genome. All other plas- tid proteins are expressed from nuclear genes. The devel- opment and differentiation of photosynthetically compe- tent chloroplasts and other plastid types thus present a challenging opportunity: to decipher how plastid gene ex- pression is controlled temporally and spatially in different plant organs, and also in coordination with the expression of nuclear genes for chloroplast proteins. Initial efforts to analyze the controls of plastid gene expression have con- centrated on the transcription of genes for photosynthetic proteins and tRNAs. Recent progress appears to support a model that places a major emphasis on posttranscrip- tional and translational regulatory mechanisms. In con- trast, known nuclear genes for photosynthetic proteins ap- pear to be regulated primarily at the level of transcription. The purpose of this review is to discuss some of the cen- tral problems and ideas in the field of chloroplast gene ex- pression, not to provide a comprehensive review on all that is known. (For further information on chloroplast ge- nome structures, genes, and transcriptional and transla- tional components, readers should consult Whitfeld and Bottomley, 1983; Ellis, 1984; Sugiura, 1987; Umesono and Ozeki, 1987; Gruissem, 1989; Mullet, 1988; Bonham- Smith and Bourque, 1988.) Linkage of Genes in Many Chloroplast Transcription Units Is Conserved Compared with the small number of genes in animal, fun- gal and plant mitochondria, the chloroplast genome con- tains a substantially larger number of genes, encoding both genetic and photosynthetic functions. The genes identified thus far include a complete set of 30 tRNAs, four ribosomal RNAs (23S, 16S, 5S, and 4.5s) and 20 ribo- somal proteins. Twenty-two genes encode proteins for thylakoid membrane complexes (photosystem I, photosys- tern II, cytochrome bsf complex, and ATP synthase), and the sequences of six other open reading frames share similarities with the mitochondrial genes for the subunits of the human respiratory chain NADH dehydrogenase. Several of the remaining unidentified reading frames are conserved between diverse species, which suggests that they may also encode functional plastid polypeptides. Most plastid genes are organized into polycistronic tran- scription units reminiscent of bacterial operons. The se- quence analysis of the entire tobacco and liverwort chlo- roplast genomes (Shinozaki et al., 1986; Ohyama et al., 1986) together with the partial sequence and mapping data from other plant chloroplast genomes, has revealed that the arrangement of genes within these transcription units is highly conserved, although transcription are extensively rearranged in some plant species (reviewed by Palmer, 1985). Detailed mapping of chloroplast DNAs from pea and geranium, for example, has found that such rearrangements involve primarily inversions of large clus- ters of genes. Most, but not all, the genes linked in these clusters are cotranscribed. It has been possible, at least some cases, to trace the linkage of chloroplast gene sets to the cyanobacterial genome (Cozens et al., 1986) which is the putative ancestral genome of chloroplast genome. However, the conserved arrangement of genes in plant chloroplast genomes is not found algae, for which Chlamydomonas and Euglena are the best studied examples, possibly indicating different endosymbiotic events. Chloroplast RNA Polymerases and Promoter Regions The possibility that transcriptional regulation chlo- roplast genes could be a key control during chloroplast development in plants spurred early investigations into the transcriptional components of this organelle. Applica- tion of different schemes for preparing DNA-dependent RNA polymerase from chloroplasts led to the intriguing idea that chloroplasts of algae and plants may contain at least two different RNA polymerase activities distinguish-

https://www.cell.com/cell/pdf/0092-8674(89)90889-1.pdf

Chloroplast Gene Expression: How Plants Turn Their Plastids On Review

Plastids are semiautonomous plant organelles exhibiting their own transcription-translation systems that originated from a cyanobacteria-related endosymbiotic prokaryote. As a consequence of massive gene transfer to nuclei and gene disappearance during evolution, the extant plastid genome is a small circular DNA encoding only ca. 120 genes (less than 5% of cyanobacterial genes). Therefore, it was assumed that plastids have a simple transcription-regulatory system. Later, however, it was revealed that plastid transcription is a multistep gene regulation system and plays a crucial role in developmental and environmental regulation of plastid gene expression. Recent molecular and genetic approaches have identified several new players involved in transcriptional regulation in plastids, such as multiple RNA polymerases, plastid sigma factors, transcription regulators, nucleoid proteins, and various signaling factors. They have provided novel insights into the molecular basis of plastid transcription in higher plants. This review summarizes state-of-the-art knowledge of molecular mechanisms that regulate plastid transcription in higher plants.

Plastid RNA polymerases, promoters, and transcription regulators in higher plants.

Two promoters, Pl and P2, have been identified upstream of the structural gene for the 16S rRNA in spinach chloroplasts (Lescure et al. 1985). A highly purified soluble spinach chloroplast RNA polymerase is shown to initiate correctly in vitro the transcription of the ribosomal DNA operon of spinach chloroplasts at the PI promoter. The P2 promoter is used only by the E. coli RNA polymerase either in vivo or in vitro. However, the P1 promoter is used at a higher frequency than the P2 promoter by the heterologous system. Both promoters appear to be used more efficiently if the DNA template is in a supercoiled form. The P2 promoter is not used in vivo in chloroplasts even if the processing of RNAs is blocked by using lincomycin.

In vitro transcription initiation of the rDNA operon of spinach chloroplast by a highly purified soluble homologous RNA polymerase

Two potential prokaryotic promoters, P1 and P2, are characterized 164 and 114 bp upstream of the spinach chloroplast 16S rRNA gene. The strengths of these promoters, calculated according to an homology score established for E. coli RNA-polymerase, are identical. Experiments performed with a Taq I-DNA fragment, containing 16 bp of the 16S rDNA and 243 bp upstream of the gene, give evidence that in vitro, E. coli RNA-polymerase starts transcription at these two promoters. These results are based on both the size of the transcripts and their nucleotide sequences. A possible regulation by differential control of these dual promoters is suggested. S1 mapping with RNAs extracted either from green or from etiolated spinach plants, indicates that, at these two steps of plastid development, transcription in vivo starts at P1. Surprisingly only P2 appears to be conserved in the homologous sequences reported for maize, mustard and Spirodela.

In vitro transcription initiation of the spinach chloroplast 16S rRNA gene at two tandem promoters

The nucleotide sequence of the spinach chloroplast 5S rRNA gene and its flanking regions has been determined. A prokaryotic type promoter is to be found upstream of the 5S rRNA gene. Northern blot experiments with selected gene probes show that the 5S gene is co-transcribed with the other ribosomal genes of the operon. This result is confirmed by 5′ S1 mapping of in vivo RNAs synthesised in chloroplasts or in an E. coli strain harboring a mutlicopy plasmid containing the 5S rRNA gene and its flanking regions. In vitro transcription experiments show that initiation of transcription does not occur at the level of the putative 5S rRNA gene promoter. Therefore, we conclude that the 5S rRNA is synthesized only be co-transcription of its gene with the other ribosomal genes of the operon. 3′ S1 nuclease mapping in the spacer region between the 4.5S and the 5S rRNA genes reveals a set of protected fragments located in an A.T rich region downstream of a very stable hairpin and immediately upstream of the putative 5S promoter. This result is interpreted by the presence of preterminated transcripts or processing sites in this region.

Structure and transcription of the 5S rRNA gene from spinach chloroplasts.

The translation machinery of chloroplast is made of specific components built up within the organelle. As it will be shown in this presentation many components of chloroplast ribosomes are encoded in the chloroplast genome albeit the largest part of ribosomal component are provided by the nucleus genome.

Cordelia Bisanz-Seyer

Papers

In vitro transcription initiation of the rDNA operon of spinach chloroplast by a highly purified soluble homologous RNA polymerase

In vitro transcription initiation of the spinach chloroplast 16S rRNA gene at two tandem promoters

Structure and transcription of the 5S rRNA gene from spinach chloroplasts.

Biosynthesis of Chloroplast Ribosomal Components