Which of the following plant cell structures is most likely to contain chlorophyll A?

Chloroplasts and Chloroplast Genomes

Chloroplasts are chlorophyll-containing organelles in plant cells; they play a vital role for life on Earth since photosynthesis takes place in chloroplasts. Chloroplasts develop from proplastids, as do chromoplasts, leucoplasts, and other plastids.

The existence of functioning DNA in chloroplasts (chloroplast DNA (cpDNA)) and other plastids is one of the main findings supporting their origin as prokaryotic (cyanobacterial) symbionts during the early evolution of life. The DNA contained in the different types of plastids of a higher plant is identical. Accordingly, photosynthesizing leaves contain not only cpDNA, but also other plant tissues, including roots and woody tissue. Compared with their cyanobacterial ancestors, chloroplasts have lost most of their genes. The size of the chloroplast genome varies between 100 and 200 kb for most plants, but both smaller and larger chloroplast genomes exist. The circular DNA of higher plants is principally structured in two inverted repeats (IRs) with reverse polarity separating a large and a small single-copy region.

CpDNA contains essential genes for the functioning of the metabolism of a plant, for example, for photosynthesis and transcription, as well as ribosomal RNA and ∼30 transfer RNA genes. However, numerous genes of the nucleus also control the structure and function of chloroplasts, which are accordingly regarded as semiautonomous cell organelles.

Abstract

Chloroplasts are the organelles specialized in carrying out the photosynthetic process, which uses light energy to synthesize organic compounds; for this reason, they are common to all photoautotrophic eukaryotes.

Besides the biosynthetic pathways directly related to photosynthesis, such as synthesis of pigments (chlorophylls and carotenoids), conversion of CO2 to carbohydrates and reduction and organization of sulfur and nitrogen, several other metabolic pathways occur in chloroplasts. These organelles produce or participate in the production of a series of essential compounds required by other cell compartments. For instance, they are the primary site of biosynthesis of fatty acids, isoprenoids, tetrapyrroles, and aminoacids, as well as of purines, pirimidines, and pentoses necessary for nucleic acid buildup. Thus, chloroplasts, in addition to photosynthesis, play other essential roles in sustaining the metabolism of the cell and the whole plant. The complexity and variety of chloroplast activities can arise from the fact that this, which is now a cell organelle, was originally an organism.

Plastids

The chloroplast belongs to the organellar class of plastids. In lower photoautotroph organisms, like algae, which do not possess cell types with very distinct functions, the photosynthetic chloroplast has remained the sole kind of plastid. On the contrary, in terrestrial plants, which evolved highly specialized tissues and organs, different kinds of plastids with specific structures and functions arose from the original chloroplast. In these plants the class of plastids, besides the green chloroplasts, includes uncolored amyloplasts, whose function is to store large amounts of starch (Figure 5) and yellow-red-brown chromoplasts which synthesize a lot of carotenoids (Figure 5) and have attractive functions. Apart from the lack of thylakoids, these nonphotosynthesizing plastids share common characteristics with the chloroplasts, like the envelope membranes, the prokaryotic DNA and the 70S ribosomes. Furthermore, they maintain several biosynthetic pathways carried out by enzymes inserted in the stroma or bound to the envelope, such as those of fatty acids and terpenoids.

All the plastids can differentiate from proplastids. However, the possibility also exists that a plastid derives from the conversion of another kind of plastid, according to the “cyclic model” (Figure 5) proposed already by Schimper at the end of 1800. These reversible plastid interconversions depend on cell developmental programs as well as on endogenous (hormones and nutrients) or environmental (light and temperature) signals. Only the chromoplasts (named gerontoplasts) derived from chloroplast degeneration during a green tissue senescence cannot undergo further conversion.

Abstract

Chloroplasts are the photosynthetic organelles of green algae and plants. Owing to their endosymbiotic origin, they contain their own genome with about 100 genes. Compared with their cyanobacterial ancestors, chloroplasts have lost most of their genes, due to either gene loss or transfer to the nucleus. Therefore, many chloroplast multiprotein complexes are of dual genetic origin with nuclear and chloroplast-encoded subunits. On the DNA level, chloroplast DNA can be subject to homologous recombination and nonhomologous end-joining, the former being exploited in plant biotechnology and the latter in the repair of double-stranded breaks. On the RNA level, some chloroplast sequences undergo editing of RNA, in particular, conversion of C to U. Like mitochondria, chloroplasts can be either inherited from both or only one parent, depending on the size of the gametes and mechanisms that specifically eliminate chloroplast DNA from one parent.

Photosynthesis

Chloroplasts are common targets of many plant viruses [(e.g., TMV, Plum pox virus (PPV), and Potato virus Y (PVY)] that are exploited for virus propagation and replication. To support the latter activities, these viruses down-regulate many chloroplast and photosynthesis-related genes, detrimentally affecting both chloroplast structure as well as the process of photosynthesis itself. Transcriptome and metabolome analyses have documented reduced photosynthesis, increased lipid peroxidation, and accumulation of reactive oxygen species (ROS). Importantly, ROS can (1) elicit pathogen restriction and often localized death of host plant cells at infection sites, and (2) act as a diffusible signal that induces antioxidant and pathogenesis-related defense responses in adjacent plant cells. Chloroplast–virus interactions also impair chloroplast division, altering consequently the normal number of chloroplasts per cell and affecting chloroplast relocation movement within the cell.

III Structure of Chloroplasts As Revealed by Light Microscopy

Chloroplasts are easily viewed in the in vivo state by light microscopy. As mentioned earlier, these organelles may assume bizarre shapes, especially in some algae. In many plants, however, the chloroplast appears as a green saucer-shaped body 5–10 µ in diameter. In green algae and some bryophytes the chloroplast contains an organized body called the pyrenoid, which is often surrounded by starch plates or lipid reserves. Chlorophyll as observed by light microscopy in the chloroplasts of algae and bryophytes appears uniformly distributed. In higher plants, however, the chloroplast from top view is seen to consist of a green field filled with small (0.2−1 µ) totally absorbing bodies called grana. The green field in which the grana lie is referred to as the stroma region of the chloroplast. Side views of the chloroplast show that the grana regions are interconnected by material indistinguishable from the grana themselves. These general observations were summarized by Heitz (9) in 1936. Two of his photographs illustrating these aspects of chloroplast morphology are reproduced in Fig. 2. Higher plant chloroplasts may be viewed by fluorescence microscopy using blue actinic light and observing the red fluorescence of chlorophyll. The chlorophyll fluorescence is seen to reside primarily in the grana stacks. Spencer and Wildman (8) have interpreted this to mean that the chlorophyll is localized in the grana regions of the chloroplast. However, we know from electron microscopy that not all membranes within the chloroplast are in the grana stacks, but that many run between grana stacks. Do these intergrana membranes contain chlorophyll? It is doubtful that fluorescence observations of whole chloroplasts will give us an answer since the electron micrographs show that the membrane concentration in the grana stack is much larger than the membrane concentration in the stroma. A similar ratio of fluorescence intensities might obscure fluorescence from the intergrana areas. Also, as mentioned in the next section, certain higher plant cells contain only large nongranal membranes and no grana membranes, though they appear to be photosynthetic. Obviously in these systems chlorophyll is distributed in the large membrane system of the chloroplast.

Some recent experiments by Lintilhac and Park (10) support the arguments that chlorophyll is uniformly distributed throughout the internal membrane system. Chloroplast internal membranes placed on an electron microscope grid were observed by both fluorescence and electron microscopy (see Fig. 3). All the membranes, both small and large thylakoids (11), are seen to contain chlorophyll. Direct obervations of this sort are contrary to the conclusions of Spencer and Wildman.

The light microscope has been used to study both dichroism and birefringence in chloroplasts. Since dichroism is considered in Chapter 11 by Butler, we are only concerned with birefringence here. Menke (12) and Frey-Wyssling (13) both studied chloroplast birefringence in media of varying refractive index. In this way they could differentiate between intrinsic and form birefringence. Form birefringence was interpreted as resulting from a layered system (12, 13) within the chloroplast. Frey-Wyssling proposed a model consisting of alternate layers of protein and lipid to account for the form birefringence. This model of layered structures was to a large extent realized with the application of electron microscopy to chloroplast structure.

Chloroplast structure and function are closely allied. For this reason it is important that the biochemist be aware of the morphological status of the chloroplasts with which he works. Initial studies by Kahn and von Wettstein (14), Spencer and Wildman (8), and Spencer and Unt (7) show that chloroplasts isolated in 0.4 M sucrose buffered with Tris or phosphate tend to be of two types. The first type retains its outer membrane and refractal jacket of stroma protein around the grana membrane and is called a class I chloroplast. The second type of chloroplast becomes ruptured during the isolation procedure and loses its outer membrane and stroma material. The latter type is referred to as a class II chloroplast. The biochemical assays by Spencer and Unt show that class I chloroplasts retain to the greatest extent the properties of in vivo chloroplasts—that is, comparatively high rates of CO2 fixation (10 µmoles/hr per milligram chlorophyll), low rates of Hill reaction due to coupled phosphorylation, and ability to form pseudopodia when resuspended in appropriate media. Heitz had shown that chloroplast pseudopodia formation was a widespread and normal occurrence in plant cells. A drawing of this phenomenon taken from Heitz's paper appears in Fig. 4, in which the pseudopodia are shown extending into the cytoplasm. These observations of Heitz have been extended by Spencer and Wildman (8) and by Wildman et al. (15). Interestingly enough, the relatively high rates of CO2 fixation in class I chloroplasts are attainable with no added cofactors. Thus it would seem that the integrity of the outer membrane has retained these cofactors in the chloroplasts, a situation that does not occur in other biochemical preparations. In conclusion, the light microscope is and continues to be a very valuable tool for studying the chloroplast in its in vivo environment. Light microscopy of chloroplast preparations is also a useful tool for the biochemist who wishes to better understand the photosynthetic capacities of his material.

6 Recovery of intact chloroplasts through Percoll

Abstract

Chloroplasts removed from their species of origin may survive for various periods and even photosynthesize in foreign cells. One of the best studied and impressively long, naturally occurring examples of chloroplast persistence, and function inside foreign cells are the algal chloroplasts taken up by specialized cells of certain sacoglossan sea slugs, a phenomenon called chloroplast symbiosis or kleptoplasty. Among sacoglossan species, kleptoplastic associations vary widely in length and function, with some animals immediately digesting chloroplasts, while others maintain functional plastids for over 10 months. Kleptoplasty is a complex process in long-term associations, and research on this topic has focused on a variety of aspects including plastid uptake and digestive physiology of the sea slugs, the longevity and maintenance of symbiotic associations, biochemical interactions between captured algal plastids and slug cells, and the role of horizontal gene transfers between the sea slug and algal food sources. Although the biochemistry underlying chloroplast symbiosis has been extensively examined in only a few slug species, it is obvious that the mechanisms vary from species to species. In this chapter, we examine those mechanisms from early discoveries to the most current research.

Section 2.3 Deducing that the chloroplasts in plant cells are captured cyanobacteria

Chloroplasts are the organelles that produce glucose and oxygen, via oxygenic photosynthesis, a process that can be loosely summarized as:

carbon dioxide + water + light energy -> carbohydrate + oxygen

Archaeplastida is the class of organisms that comprises all plant life on the planet. Every species of Class Archaeplastida contains chloroplasts or has an ancestral species that contained chloroplasts. Aside from photosynthesis occurring in plants, we can also observe photosynthesis in cyanobacteria. It has been inferred that plants acquired photosynthesis when they engulfed photosynthesizing cyanobacteria that evolved into self-replicating chloroplasts. This inference is based on the following observations: [Glossary Cyanobacteria]

That chlorplasts are self-replicating.

Because chloroplasts, like mitochondria, self-replicate, we infer that they were formerly free-living organisms in their own right. We eukaryotes play host to a variety of organisms that replicate inside our cells (e.g., infective mycoplasma, viruses, and infective apicomplexans), but mitochondria and chloroplasts are the only intracellular self-replicating structures that provide vital services to host cells and that are always passed through the germ line via the cytoplasm of oocytes or, in the case of plants, gametophytes (Fig. 2.7). [Glossary Eukaryote]

The biochemical machinery of chloroplasts is nearly identical to that of its presumed bacterial precursor.

Oxygenic photosynthesis required the independent evolution of two different pathways that combine to complete the full biosynthetic cycle.8 The first pathway involves the capture of photons to hydrolyze water, yielding oxygen, NADPH and ATP. The second, light-independent pathway, is the Calvin cycle, driven by high-energy molecules (NADPH and ATP) created by the first pathway to form simple sugars from carbon dioxide. The achievement of photosynthesis was such an unlikely happenstance that we would expect to encounter de novo evolutions of photosynthesis only rarely, if at all. In fact, in the bacterial kingdom, we see only one clear-cut example of an organism achieving oxygenic photosynthesis: the cyanobacteria. When the cyanobacteria evolved oxygenic photosynthesis, about 2.5 billion years ago, they launched a terraforming project that eventually changed the atmosphere of our planet from an anoxic environment to an oxygen-rich environment. In so doing the cyanobacteria ordained the extinction of an untold number of anaerobic species. [Glossary Anaerobic]

When we examine the photosynthesis pathways in plant chloroplasts and the photosynthesis pathways in cyanobacteria, they are essentially identical. Hence, it seems likely that the photosynthetic pathway of plant cells was provided by cyanobacteria, which predate the first eukaryotes.

As an aside, there is some evidence to suggest that we may be currently witnessing a repeat of the process by which a primitive eukaryote stole a cyanobacteria, imitating Class Archaeplastida. Paulinella chromatophora, a member of the eukaryotic class Rhizaria, seems to have captured its own cyanobacteria and created its own permanent chloroplast-like organelle.9

Chloroplasts are wrapped by two membranes. All other plant organelles are wrapped by one membrane.

From whence did the second membrane of chloroplasts come? One layer is believed to have been contributed by the captured cyanobacteria, and one layer presumably came from the original plant cell that captured the cyanobacteria. The two-membrane chloroplast, observable under the microscope, is a key piece of evidence strengthening the theory that chloroplasts evolved when a eukaryotic cell captured a cyanobacteria and established Class Archaeplastida, the kingdom of plants.

The chloroplasts of non-Archaeplastida eukaryotes have three or four membrane layers.

Chloroplasts are found in a variety of eukaryotic species that are not members of Archaeplastida, the ancestral class of plants. How did nonplant species acquire an organelle that evolved as a characteristic feature of the plant kingdom? It seems that the nonplant organisms that contain chloroplasts simply stole their chloroplasts from other organisms, particularly plants. Whereas the chloroplasts of plants always have exactly two membranes, the chloroplasts of nonplant organisms have three or four membrane layers, suggesting that an organism of Class Archaeplastida was engulfed, and the double membranes of the chloroplast plus the membranes of the engulfing organisms were entrapped in the process.

Is there any reason to think that organisms go about stealing organelles from other organisms? It seems that plant chloroplasts can be acquired temporarily through a process called kleptoplasty. The kleptoplastic cell captures a chloroplast from an algae and uses the captured chloroplast for a short period (a few days to a few months) until the chloroplast degenerates. These chloroplasts are not self-replicating. When new chloroplasts are needed, the kleptoplastic organism simply ravages another colony of algae. The sacoglossan sea slug has achieved a photosynthetic lifestyle, all thanks to kleptoplasty. [Glossary Kleptoplast]

Sun versus shade chloroplasts

Chloroplast development is light-dependent (Apel et al., 1983) and different ultrastructures and physiologies can result as a consequence of the light environment during development (Lichtenthaler et al., 1981). Because light is limiting in the shade, chloroplasts in leaves that develop in the interior of a tree canopy develop to favor the light-dependent reactions over the light-independent reactions. Shade-type chloroplasts are optimized for light capture with larger grana, more thylakoids per granum and more chlorophyll per antenna complex. Stromal volume is concomitantly reduced. Sun-type chloroplasts, which develop at the exterior of the canopy where light is not limiting, have smaller grana, less chlorophyll (reduced capacity for the light-dependent reactions) and more stromal volume (increased capacity for the light-independent reactions). Both chloroplast types can be found on the same plant, thus these two different developmental outcomes are driven solely by the light environment experienced during leaf expansion.