The light-independant reactions of photosynthesis occur in the stroma of the chloroplast and involve the conversion of carbon dioxide and other compounds into glucose. The light-independent reactions can be split into three stages, these are carbon fixation, the reduction reactions and finally the regeneration of ribulose bisphosphate. Collectively these stages are known as the Calvin Cycle.
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.
Students also investigate the properties and the importance of chlorophyll. Using paper chromatography, students find that the color in green plants consists of several chlorophyll pigments. Students explore the cellular structure of plant cells to observe chloroplasts in various parts of an Elodea plant. Students connect the role of chloroplasts to the process of photosynthesis. From these observations, students conclude that photosynthesis takes place in Elodea.
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.
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).
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.
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.
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).
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.
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.