Chloroplast

A distinctive feature of chloroplasts of plants and algae is their extensive, internal, green, chlorophyll-containing membrane system, called thylakoid membranes, where the primary reactions of photosynthesis occur. This system of photosynthetic reaction centers converts light energy into chemical energy, which is used to drive cellular metabolism. Besides their important role in photosynthesis, chloroplasts are also involved in several biochemical pathways, such as the biosynthesis of amino acids, fatty acids, tetrapyrroles including chlorophyll and heme, carotenoids, isoprenoids and pyrimidines. Chloroplasts are also involved in carbon metabolism and in nitrogen and sulfur assimilation (1). Like mitochondria, chloroplasts possess their own genetic system, which cooperates closely with the nucleus in biosynthesizing numerous organellar components. Chloroplasts represent one type of plastid derived from colorless proplastids in the meristematic cells of plant leaves and shoots, which have only a rudimentary internal membrane system (1). Light profoundly affects the development of proplastids. They differentiate into chloroplasts in the presence of light, whereas in its absence they differentiate into etioplasts, which lack chlorophyll and contain a prolamellar body. Upon subsequent illumination, the prolamellar body gives rise to lamellae of the thylakoid membrane. Depending on the plant tissues, the developmental stage, and the environmental conditions, proplastids also differentiate into chromoplasts in petals or fruits, into leucoplasts in roots, or into amyloplasts in tubers in which starch is accumulated. Proplastids also develop into elaioplasts in glands, certain fruits and seeds, where they are involved in synthesizing lipids, terpenoids, carotenoids, and carbohydrates. Although these various plastid forms have rather distinct morphologies, plastid differentiation is reversible to a large extent, because chloroplasts develop from leucoplasts or amyloplasts, and viceversa. During transitions from chloroplasts to the other plastid forms, the expression of most organellar genes is reduced, whereas specific nuclear genes encoding plastid proteins are activated (1, 2). An important point is that all plastid types

contain an internal membrane system that is crucial for their interconversion.

1. Thylakoid Membranes and the Photosynthetic Apparatus

 The internal thylakoid membrane system consists of appressed and non-appressed flattened membrane vesicles, called grana and stroma lamellae, respectively (Fig. 1). The primary reactions of photosynthesis are catalyzed by four major protein-pigment complexes of the thylakoid membrane: (i) photosystem II and (ii) photosystem I, and their associated chlorophyll antennae, (iii) the cytochrome b6/f complex, and (iv) the ATP synthase (Fig. 1). Briefly, light energy is captured by the antennae and channeled to the reaction centers of photosystem II and photosystem I. The energy is used to energize an electron in chlorophyll and to create a stable charge separation across the membrane. This triggers a series of oxido-reductions along the photosynthetic electron-transfer chain. At one end of this chain, water is oxidized by photosystem II with concomitant evolution of oxygen and release of protons into the lumen. Then electrons are transferred to plastoquinone, to the cytochrome b6/f complex, which acts as a proton pump, and to the soluble electron carrier plastocyanin in the thylakoid lumen. At the other end of the chain, photosystem I oxidizes plastocyanin upon light absorption and transfers electrons to ferredoxin and then to NADP to form NADPH. The resulting pH gradient is used by the fourth complex, ATP synthase, to produce ATP on the stromal side. This enzyme also functions in the opposite direction by hydrolyzing ATP to pump protons into the thylakoid lumen and thus generate a pH gradient. Because the abundance of the thylakoid membrane complexes facilitates their biochemical analysis and because the state of the redox cofactors is monitored readily by spectroscopic techniques, the thylakoid membrane has been studied intensively and represents one of the best-studied membrane systems.

Figure 1. Photosynthetic complexes in the thylakoid membrane of chloroplasts. PSII (photosystem II) is located within the appressed grana region, whereas PSI (photosystem I) is located within the nonappressed stroma lamellae. The photosynthetic electron transfer chain is shown starting with water as electron donor to PSII, to plastoquinone (PQ), to the cytochrome b6/f complex (cytb6/f), to the soluble electron transfer protein plastocyanin (PC), to PSI, to ferredoxin (Fd), to ferredoxin-NADP oxidoreductase (FNR), and to NADP as final electron acceptor. Electron flow is coupled to proton translocation into the lumen. The resulting pH gradient across the thylakoid membrane drives ATP synthesis. Both ATP and NADPH are used for CO₂ fixation.

Each of the four photosynthetic complexes contains numerous protein subunits, some of which are encoded by the chloroplast genome, whereas others are encoded by the nuclear genome (Table 1; Fig. 2). The two principal reaction center polypeptides of photosystems I and II are highly hydrophobic and contain 11 and 5 transmembrane a-helices, respectively, to which most of the redox cofactors and several chlorophylls are bound with an asymmetrical distribution across the thylakoid membrane. This asymmetry is crucial for the vectorial electron transport in the membrane.

The distribution of these complexes is unequal between the appressed (grana) and nonappressed thylakoid membrane regions (3). Photosystem II is localized predominantly in the grana regions, whereas photosystem I and the ATP synthase complex are found exclusively in the nonappresssed regions. The cytochrome b6/f complex is present in both the grana and nonappressed regions. Destacking and restacking of thylakoid membranes are induced experimentally by decreasing and then increasing again the cation concentration. Remarkably, the lateral segregation of the photosynthetic complexes between grana and stromal membranes is lost upon destacking because of random mixing, but it is restored upon restacking the membranes (4). 

Figure 2. Biosynthesis of the photosynthetic apparatus and protein traffic in the chloroplast. Photosynthetic complexes consist of nucleus- and chloroplast-encoded subunits. The former are synthesized as precursors on cytosolic 80S ribosomes and targeted to the chloroplast. Upon import into the organelle, the N-terminal stromal transit peptide domain is cleaved, and the protein is directed to the stroma, to the envelope, or to the thylakoids. In the latter case, the protein contains an additional cleavable thylakoid targeting domain. Several posttranscriptional steps in the chloroplast, such as RNA stability, processing, splicing, editing, and translation, plus the assembly of protein complexes, require the action of numerous nucleus-encoded factors. Chlorophyll, the major pigment of the thylakoid membrane is synthesized entirely in the chloroplast. Synthesis starts from d-aminolevulinic acid (ALA) and involves several steps in common with heme biosynthesis until protoporphyrin IX (proto IX). One of the last steps of chlorophyll synthesis, conversion of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), requires light in land plants. Chlorophyll synthesis is tightly coordinated with the synthesis of its apoproteins. Expression of nuclear genes of photosynthetic proteins is strongly stimulated by light. Some of the chlorophyll precursors influence, directly or indirectly, expression of nuclear genes involved in photosynthesis.

Table 1. Informational Content of Chloroplast DNA from Land Plants and Green Algae

^a The number of chloroplast-encoded subunits of photosynthetic complexes. The numbers in parenthesis refer to the number of nucleus-encoded subunits.

^b These genes are found only in the chloroplast genomes of land plants.

^c The number of chloroplast genes involved in light-independent chlorophyll synthesis and CO₂ uptake. These genes are found only in the chloroplast genomes of gymnosperms, liverwort, and Chlamydomonas.

^d This gene is involved in CO2 uptake.

Other dynamic changes in thylakoid membrane organization occur when plants and algae are subjected to light of different wavelengths that is preferentially absorbed by either photosystems II or I. Under these conditions, part of the chlorophyll antenna is displaced from one photosystem to the other, so as to achieve balanced light absorption and hence optimal functioning of the two photosystems (5). Thus when photosystem II is preferentially activated, the plastoquinone pool is reduced. This leads to the activation of a protein kinase associated with the cytochrome b6/f complex, to the phosphorylation of the light-harvesting complex (LHCII), and to the concomitant movement of part of the photosystem II antenna to photosystem I. If photosystem I is preferentially activated, LHCII is dephosphorylated, and it returns to photosystem II in the grana regions.

1.1. Stroma and Carbon-Fixation Cycle 

The ATP and NADPH produced by the primary light reactions of photosynthesis are used as sources of energy and reducing power to drive the reactions of the carbon fixation cycle, which convert CO₂ into glyceraldehyde 3-phosphate, a precursor to sugars, amino acids, and fatty acids. Although these reactions are also called the “dark reactions,” the enzymes involved are inactivated in the dark and need to be reactivated by light through the reducing power generated by photosynthesis. The key reaction, which involves converting one atom of inorganic carbon, as CO₂, into organic carbon, is catalyzed by the enzyme ribulose 1,5 bisphosphate carboxylase (Rubisco), a large stromal enzyme that works only sluggishly (6). Therefore, it is required in large amounts and is thought to be the most abundant protein on earth. This enzyme also has an oxygenase activity, which predominates if the concentration of CO₂ is low. Under these conditions it catalyzes the first step of a pathway called photorespiration, which ultimately liberates CO₂ and thereby reverses the photosynthetic reaction. In addition, the stroma includes a large number of proteins involved in several important metabolic pathways (amino acid and fatty acid synthesis, sulfur and nitrogen assimilation). The chloroplast transcription and translation systems are also contained in this compartment.

1.2. Chloroplast DNA and Its Informational Content 

Chloroplasts together with mitochondria, are the only cellular organelles containing their own apparatus for protein biosynthesis. It consists of chloroplast DNA, RNA polymerase, enzymes involved in RNA metabolism, ribosomes, transfer RNA, and several translation factors. Chloroplast ribosomes resemble those of bacteria and have similar ribosomal RNA and proteins and sensitivity to a similar spectrum of antibiotics. The circular chloroplast DNA molecules range in size between 70 kb and 400 kb (7) and are present in about 100 copies per chloroplast. A typical mesophyll cell contains close to 100 plastids and thus about 10,000 chloroplast DNA circles. A dozen chloroplast genomes from several vascular plants and algae have been sequenced. These sequences have revealed the existence of about 120 chloroplast genes in plants and green algae. They include about 50 genes that encode components of the transcriptional apparatus (subunits of RNA polymerase) and of the translational apparatus (ribosomal RNA, ribosomal proteins, transfer RNA, and translation factors). About 40 genes are involved in photosynthesis, and they encode some of the subunits of photosystems I and II, the cytochrome b6/f complex, ATP synthase and Rubisco (see Table 1). The other subunits of these complexes are encoded by the nuclear genome, translated on cytosolic ribosomes, and imported posttranslationally into the chloroplast. The genes involved in the plastid protein synthesizing system and in photosynthesis have been conserved during evolutionary divergence of the chloroplast genomes of plants and green algae. The remaining chloroplast genes, however, have not been universally conserved. Eleven genes encoding subunits of NADH dehydrogenase are present in the chloroplasts of plants, but not in algae. Whereas the role of the mitochondrial NADH dehydrogenase in respiration is well understood, the function of the chloroplast enzyme has not yet been elucidated. It could be involved in a chlororespiratory pathway by reducing the plastoquinone pool in the dark, which is ultimately oxidized by molecular oxygen via unknown redox components (8).

In most plants, one of the last steps of the chlorophyll synthesis pathway, the conversion of protochlorophyllide into chlorophyllide, is light-dependent. In green algae and gymnosperms, an alternative light-independent pathway for chlorophyll synthesis is mediated by three chloroplast genes that are absent in angiosperms. The sequences of chloroplast genomes have revealed additional genes whose functions are still unknown.

The chloroplast genomes of nongreen algae contain twice as many genes as those of higher plants. Additional genes include those required for photosynthesis that are nucleus-encoded in plants and green algae, genes involved in the synthesis of fatty acids, amino acids, and pigments, genes required for protein folding and transport, and additional genes of unknown function (9). The smallest plastid genome identified, that of the white parasitic plant Epifagus virginiana, is only 70 kbp in size. It has lost all the genes involved in photosynthesis, and the remaining genes encode mostly components of the plastid protein synthesizing system.

It is generally admitted that plastids originated as the result of an endosymbiotic event in which a prokaryotic photosynthetic organism, probably similar to a cyanobacterium, invaded a primitive eukaryotic cell. Strong support for this endosymbiotic hypothesis arises from the considerable similarity between the transcriptional and translational systems of prokaryotes and plastids. It is  thought that during evolution genetic information from the intruder was gradually lost and transferred to the nucleus of the host. The question thus arises why chloroplast DNA has been maintained. One possibility is that this evolutionary plastid genome size reduction is still in progress and has not yet reached its final stage. Another possibility is that the plastid protein synthesizing apparatus is essential for synthesizing the large hydrophobic polypeptides of the photosynthetic reaction centers, which cannot be translocated across the plastid envelope membrane. A third recently advanced hypothesis is that the presence of the plastid protein synthesizing system is essential to allow a rapid response of plastid gene expression to environmental changes (10.(

1.3. Chloroplast Gene Expression 

Two distinct RNA polymerases are present in the chloroplasts of higher plants. One is similar to its bacterial homologue, and its subunits are encoded by chloroplast genes. This enzyme transcribes primarily genes involved in photosynthesis, which are expressed at a high level. The second plastid RNA polymerase is nucleus-encoded and is required for expressing the nonphotosynthetic plastid functions necessary for plant growth (11). Many chloroplast genes are organized in large transcription units. These units are transcribed into large precursor transcripts, which then are processed into individual messenger RNA (mRNA) molecules. Chloroplasts contain RNA splicing systems, because several plastid genes contain introns, mostly group II and group I, which have a characteristic secondary structure (12). These introns have also been found in mitochondrial genes, and some of them are self-splicing. Splicing in the chloroplast is rather complex, as in the case of the psaA gene encoding one of the reaction center polypeptides of photosystem I in the green alga Chlamydomonas. This gene consists of three coding regions (exons) that are widely separated on the chloroplast genome and are flanked by group II intron sequences (13). They are transcribed individually, and maturation of the psaA mRNA depends on two trans-splicing reactions in which the separate transcripts of the three exons are spliced together. A particularly intriguing feature is that one of the introns is split into three parts (14). This has interesting evolutionary implications because it is thought that group II introns represent the precursors of nuclear introns and their associated splicing factors. In this view, the split chloroplast intron may represent an intermediate between group II and nuclear introns. The chloroplast genetic system has evolved at a rather slow rate and could have therefore maintained some ancient gene organization.

Another unusual feature of chloroplast RNA metabolism is RNA editing in vascular plants (15). Editing in chloroplasts is a posttranscriptional process in which specific C residues of a primary transcript are changed to U. Editing has important implications for interpreting DNA genomic sequence data. As an example, an ACG triplet may be edited to AUG, thereby creating a new initiation codon, which could not be identified in the DNA sequence. Alternatively, an editing event may change an internal codon and thus change the corresponding amino acid predicted by the DNA sequence. Therefore, sequencing of chloroplast genomes may not allow identifying of all of the plastid genes.

Because the subunits, redox cofactors, and pigments of photosynthetic complexes are synthesized by two distinct genetic systems, the process has to occur in a coordinated way (Fig. 2). Genetic studies with Chlamydomonas and maize have indeed revealed the existence of highly complex interactions between nucleus and chloroplast (16). A large number of nuclear genes are involved in chloroplast gene expression. They encode factors targeted to the chloroplast that act at different posttranscriptional steps, such as RNA processing, RNA stability, RNA splicing, translation, and the assembly of photosynthetic complexes. Light strongly enhances some of these steps, especially translation. Several translational activators have been identified, which act at the level of initiating translation. Translation in the chloroplast occurs on chloroplast ribosomes, which are often closely associated with the thylakoid membrane. Cotranslational insertion into the thylakoid membrane has been proposed for the hydrophobic reaction center polypeptides. In addition, synthesis of chlorophyll and its apoproteins needs to be strictly coordinated, because free chlorophyll is highly photoreactive and causes serious damage to the cell.

1.4. Chloroplast-Nuclear Cross talk 

Chloroplast function and development depend to a large extent on the nucleus. A large number of nuclear genes encode chloroplast structural components and enzymes and are involved in regulating chloroplast gene expression. Reciprocally, chloroplasts also influence nuclear gene activity. This is apparent in mutant plants with defective chloroplasts, where nuclear genes of proteins involved in photosynthesis are no longer expressed. As an example, when carotenoid synthesis is inhibited, chloroplasts rapidly bleach in strong light because chlorophyll is photooxidized in the absence of carotenoids (17). Under these conditions, expression of nuclear genes that code for several abundant chloroplast proteins involved in photosynthesis is specifically repressed. A block in chloroplast protein synthesis has a similar effect (18). These observations imply the existence of a plastid-derived factor that directly or indirectly influences nuclear gene activity. There are mutants of Arabidopsis in which the transduction of this plastid-derived signal to the nucleus is affected (19). The nature of the plastid factor is still unknown in plants, although studies with Chlamydomonas suggest that some porphyrin compounds, which act as intermediates in the chlorophyll biosynthetic pathway, are involved in this response (Fig. 2, 20).

1.5. Protein Sorting in the Chloroplast 

Chloroplasts are bounded by an envelope that consists of the outer and inner membranes. The outer membrane is freely permeable to ions and small molecules, whereas the inner membrane is highly selective and contains specific translocators and permeases that allow regulated metabolic transport between cytosol and stroma. The envelope also contains the protein import system.

 From just the modest size of the plastid genome, it is clear that the majority of the chloroplast proteins are encoded by nuclear genes and imported into the chloroplast. Six chloroplast compartments can be distinguished: (1) the outer envelope membrane, (2) the intermembrane space, (3) the inner envelope membrane, (4) the stroma, (5) the thylakoid membrane, and (6) the lumen. Nucleus-encoded proteins destined to the chloroplast are synthesized as precursor proteins containing, in most cases, a transient N-terminal transit peptide (21). Transit peptides are both necessary and sufficient to import a polypeptide into the chloroplast. Transit peptides of stromal proteins consist of 30 to 120 residues in only a poorly conserved sequence. The only distinguishing feature is that they are rich in hydroxylated amino acids and deficient in acidic residues. Recognition of the protein import receptor by the transit peptide is followed by translocation of the precursor protein in an extended conformation across the two envelope membranes. ATP and GTP are the sole energy sources for this process, which also requires the participation of several factors to unfold protein on the outside and to refold protein on the inside of the organelle. Several molecular chaperones play an important role in the proper folding of the polypeptides that enter the chloroplast (21) .Translocation of the precursor of protochlorophyllide oxidoreductase, an enzyme involved in the last step of chlorophyll synthesis, also requires the presence of its substrate, protochlorophyllide, inside the plastid (22). This raises the possibility that the substrate drives the translocation by inducing or stabilizing folding of the enzyme on the stromal side of the envelope.

Thylakoid precursor proteins contain a bipartite transit peptide. The first domain targets the protein to the stroma, and the second hydrophobic domain, which resembles the signal sequences of secretory proteins, acts as the thylakoid targeting domain. Surprisingly, there are four pathways for protein translocation into or across the thylakoid membrane (21). The first corresponds to the bacterial protein secretion system and uses Sec proteins homologous to the bacterial SecA and SecY proteins. The second uses a system involving a signal recognition particle. The third pathway is rather unique because it uses only the trans-thylakoid pH gradient as an energy source. The fourth pathway involves spontaneous insertion of certain proteins into the thylakoid membrane.

 Insertion of proteins into the chloroplast envelope occurs by several routes. Some nucleus-encoded polypeptide chains lack a cleavable transit peptide and are inserted directly into the outer and inner membranes. Other envelope membrane proteins containing a cleavable transit peptide use the general import pathway. At least one inner membrane envelope protein is encoded by the chloroplast genome, so it must contain an appropriate targeting signal.

1.6. Chloroplast Engineering 

A major breakthrough in chloroplast research in 1988 was the development of an efficient method for genetically transforming chloroplasts of the green alga Chlamydomonas (23), which was subsequently adapted to higher plants (24). In this method, tungsten or gold particles are coated with DNA and bombarded into cells with a particle gun. Upon entry of the particles into chloroplasts, the DNA is released and integrated into the chloroplast chromosome by homologous recombination. The existence of an efficient chloroplast homologous recombination system and the development of selectable markers for chloroplast transformation have opened the door to manipulating the chloroplast genome. In particular, this new technology allows directed chloroplast gene disruption, a powerful tool for elucidating the role of genes of unknown function. It has also permitted site-directed mutagenesis of specific residues of photosynthetic reaction center polypeptides so as to gain new insights into their structure-function relationship, and it has been very useful for studying chloroplast gene expression. Chloroplast transformation has important applications for plant biotechnology and protein engineering. Because the chloroplast genome is present in multiple copies, up to 10,000 per cell, new genetic information introduced into plastids is amplified. In principle, this opens the possibility of expressing foreign proteins of commercial interest in large quantities. The expression of foreign genes in the chloroplast compartment offers the additional advantage of considerably reducing the risk of transfer of new genetic material to the environment because the chloroplasts from the male parent are not transmitted to the progeny in most crop plants.

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