During the evolution of higher plants, a necessity arose in that plants needed to attract insects and mammals to them in order to facilitate flower pollination and to aid in the dispersal of seeds within fleshy fruits. In order for higher plants to be prominent visually within the flora, the development of brightly coloured structures occurred, primarily in the petals of flowers and in the tissues of fleshy fruits. The accumulation of pigments within the plastids in these tissues led to the formation of a distinct type of plastid, namely the chromoplast. Most of the pigments that are laid down in chromoplasts are carotenoids, which are synthesised from the C40 molecule phytoene, and constitute several different types, namely carotenes, lycopene, lutein, violaxanthin, and neoxanthin (Camara et al. 1995; Cunningham and Gantt 1998; Bramley 2002). These classes of molecules are not the sole preserve of chromoplasts, since several are commonly found on thylakoid membranes in the chloroplast where they function as accessory pigments in light capture and energy dissipation. In addition, the carotenoid- related pigment astaxanthin is the basis of the red-pink colouration in several animals including flamingo, lobster, and shrimp (Armstrong and Hearst 1996). Other types of soluble pigments, which are found in the cell's vacuole, also contribute to colouration of plant parts and in many cases a mixture of pigment types is present (Kay et al. 1981; Weston and Pyke 1999).
Detailed structural analysis of chromoplasts in different tissues and species shows great heterogeneity in their structure, which probably reflects differences in the profile of coloured pigments present. There have been efforts to categorise chromoplasts into distinct types according to the types of storage structures present within the chromoplasts, i.e. globular, membranous, or crystalline (Thomson and Whatley 1980; Camara et al. 1995). Although laudable, such a classification system can be difficult to apply to the vast range of chromoplast types found throughout nature in different tissues in different plant species. Knowledge of chromoplast biogenesis has been gained largely from a detailed study of agriculturally important fleshy fruits, primarily in tomato (Lycopersicon esculentum) (Fraser et al. 1994; Camara et al. 1995; Cunningham and Gantt 1998), and bell pepper (Hugueny et al. 1995a). During the formation of pigmented chromoplasts from green chloroplasts in unripe fleshy fruit, a controlled breakdown of chlorophyll and the thylakoid membrane occurs concurrent with a significant increase in carotenoid pigment biosynthesis. Increased expression of the ELIP gene is associated with the chloroplast to chromoplast transition and may play a role in the regulated breakdown of the extensive thylakoid membranes of the chloroplast (Bruno and Wetzel 2004). Associated with increased carotenoid biosynthesis is the upregulated expression of several nuclear genes, which are required for chromo-plast differentiation (Lawrence et al. 1993, 1997; Summer and Cline 1999). Plastid DNA appears to play a minor role in chromoplast differentiation and there is increased methylation of plastid DNA in chromoplasts (Kobayashi et al. 1990). Exactly how a chromoplast differentiation pathway is initiated in green chloroplasts in ripening fruit is unclear, even though a significant amount is known about the basic biochemistry and molecular biology of fruit ripening and the role of the hormone ethylene (Alexander and Grierson 2002). It could be argued that the chromoplast is no more than a bag into which carotenoid pigment is loaded and indeed increased transcription in a variety of carotenoid biosynthetic enzymes is a key phase in chromoplast biogenesis. Increases in enzyme activity of phytoene synthase and phytoene desaturase (Fraser et al. 1994), 1-deoxy-D-xylulose 5-phosphate synthase (Lois et al. 2000) and a plastid terminal oxidase associated with phytoene desaturation (Josse et al. 2000) are observed as chloroplast differentiate into chromoplasts. There are also increases in other proteins not associated with carotenoid metabolism and which could be best viewed as chromoplast specific differentiation genes. These include enzymes in response to oxidative stress (Livne and Gepstein 1988; Romer et al. 1992), and carotenoid sequestration proteins including fibrillin (Vishnevetsky et al. 1999).
Surprisingly the cell biology of chromoplast differentiation has been poorly described and until recently was dependent upon electron microscopy descriptions of their structure (Harris and Spurr 1969a, 1969b; Thomson and Whatley 1980; Bathgate et al. 1985). Internally, chromoplast structure appears dependant on which type of carotenoids are sequestered within them since the internal architecture is highly variable and can consist of either plastoglobules of pigment, crystalline structures of carotenoids, microfibrillar structures with sequestered carote-noids, extensive internal membranous structures or a mixture of these. Gunning (2004) shows particularly beautiful colour images of red and yellow chromoplasts in a variety of petals and fruits.
In recent years, the exploitation of green fluorescent proteins targeted to the plastid compartment has enabled chromoplasts to be observed within the whole cell and some aspects of their cell biology have been revealed, primarily in the ripening fruits of tomato (Fig. 1). In the light microscope, these red tomato chro-moplasts appear as small heterogeneously shaped organelles with little clear structure. The production of thin membranous tubules from the chromoplasts called stromules, a feature of plastids in general, has been well studied and will be discussed in Section 6. Occasionally, the membrane of tomato chromoplasts is distorted by long thin crystals of lycopene (Pyke and Howells 2002). Mature pericarp cells in the fleshy part of the ripe tomato fruit are large and may contain up to 2000 red pigmented chromoplasts, which are generated from populations of dividing chloroplasts, that accumulate during the green phase of fruit development. During the differentiation of chromoplasts from chloroplasts, a heterogeneous array of small bodies within the cell can be observed, some of which appear to be broken pieces of stromule or even vesicles which appear to bud from the chloroplast body and are revealed by the GFP they carry (Waters et al. 2004; Forth and Pyke 2006). Thus, two different processes could give rise to large populations of differentiated chromoplasts within the cell and gives support to the idea that chromoplasts are little more than storage sacs with high levels of carotenoid bio-synthetic enzyme activity.
Fig. 1 (overleaf). Variation in plastid morphology. (A) Chloroplasts in a leaf stomatal guard cells containing GFP, which fluoresces green on a background of red chlorophyll fluorescence. These chloroplasts have a conventional chloroplast morphology and have only small stromule protrusions. (B) Isolated giant mesophyll cell chloroplasts from Arabi-dopsis leaves expressing antisense copies of the FtsZ plastid division protein. These chloro-plasts are highly variable in shape but maintain their complex morphologies when isolated from the cell. (C) Plastids in the hypocotyl cells of a tobacco seedling illuminated by targeting green fluorescent protein to the plastids. These plastids show extensive stromules and complex looping. (D) Plastids in the epidermal cell of an Arabidopsis root illuminated by targeting green fluorescent protein to the plastids. These plastids are highly variable in morphology, at the most extreme showing thin stromules. (E) Image of a pericarp cell in a tomato fruit at the onset of chromoplast differentiation from chloroplasts. Green fluorescent protein has been targeted to the plastids. Yellow plastid bodies exhibit both green fluorescent protein fluorescence as well as red chlorophyll fluorescence. Some red plastid bodies contain little GFP. A large number of plastid-derived structures, which contain bright green GFP are visible, both as stromules and distinct vesicle-like structures which appear to bud off from the main plastid bodies and lack chlorophyll. (F) Two chromoplasts in a tomato pericarp cell, which contain extensive GFP in the main chromoplasts bodies and are connected by two long thin stromules which show significant beading. (G and H) The cytoplasm of a pericarp cell from a ripe tomato view with brightfield (G) and with GFP fluorescence targeted to the plastid. A stromule emanating from a plastid body is obvious as are crystals of lycopene, which contain GFP and presumably are surrounded by a chromoplast membrane.
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