The study of metabolic networks and feedback response to developmental and environmental conditions involves the profiling of natural products. In this context, phytosterolomic research can be defined as the complete identification and quantification of all sterols present in a specific biological sample. Phytosterol fingerprinting is another aspect of phytosterolomics where the unusual sterol composition of a pathogen can serve as a signature lipid for disease such as for immunocompromised patients harboring pneumocystis (Kaneshiro et al., 2002; Zhou et al., 2002). Phytosterol profiling involves techniques such as thin layer chromatography (TLC) and high-performance liquid chromatography (LC) mass spectroscopy (LC/MS) and gas chromatography mass spectroscopy (GC/MS) where individual components are separated and tentatively identified and quantified by one set of techniques with final characterization of structure determined by 1H or 13C NMR spectroscopy (Kalinowska et al., 1990; Nes et al., 1998a, 1999, 2003). HPLC equipped with a diode array detector to monitor UV is useful in the identification of the type and amount of conjugation in the sterol molecule (Nes et al., 1985; Norton and Nes, 1991; Xu et al., 1988). Separation factors in GLC and HPLC based on the retention of cholesterol relative to the retention time of sterol standards have been reported to enable the prediction of the identity of an unknown sterol (Xu et al., 1988). High-throughput screening of phytosterol mixtures by conventional chromatography and spectroscopy has not yet progressed to the point where a single method can establish the identities of a complicated set of structurally similar phytosterols. Although the sterol patterns of many plant extracts deduced by GLC are rather simple, usually consisting of three or four major sterols, the sterol composition of specific plant parts or the type of sterol in primitive versus advanced plants can be strikingly different (Nes, 1990).
The sterol side-chain can be modified by the addition of one or two supernumerary carbon atoms at C-24 with either α- or β-chirality. Establishing the stereochemistry of the 24-alkyl group of individual sterols is a formidable task and can be achieved by identifying specific side-chain carbons by high-field NMR (Fig. 9.7, Panels A–C). The assignments of the signals from the 1H or 13C NMR spectra of 1H- or 13C-labeled sterols are diagnostic for the origin of the methyl group at C-24 and the biosynthetic stereospecificity of phytosterol sidechains (Guo et al., 1996, 1995; Popják et al., 1977). The relative proportion of α- to β-isomer of a 24α/β-methyl cholesterol mixture can also be quantified by reversedphase HPLC (Guo et al., 1995) (Fig. 9.7, Panel D) (Parker and Nes, 1992). The ratio of these diastereoisomers can change as plants evolve from less to more advanced (Nes et al., 1977).
Using the modern methods of sterol analysis, it has been possible to show that phytosterols appear in all cells at all stages of development, but the type and amount of sterol is greatly influenced by ontogeny and speciation, and consequently seems to be a carefully regulated process (Nes, 1990). In spite of findings that show variability of the sterol side C-24 alkyl structure occurs widely, suggesting some sort of association between sterol structure and plant biology, relatively little is known about individual sterols or sterol sets and their role in plant physiology. As many as 60 sterols have been reported in a vascular plant, Zea mays (Guo et al., 1995), which is about one-third to one-half the number of phytosterols found in living systems; 39 sterols have also been identified in an ascomycetous fungus, Gibberella fujikuroi (Nes et al., 1989a).
The ratio of 4,4-dimethyl and 4-desmethyl sterol intermediate to 4-desmethyl sterol end product, easily determined by TLC, can be as much as 1:3 in pollen, 1:3 in the seed, and 1:9 in vegetative parts (Heupel et al., 1986; Marshall et al., 2001; Nes and Schmidt, 1988; Nes et al., 1991c). These differences in intermediate to end product ratios suggest that the phytosterol pathway is undergoing significant changes during development. Consistent with this observation, as plants mature from seed to flower the amounts of total sterol in individual parts and various cell types differ dramatically. For example, in sunflower the sterol levels are reported to be (Nes, 1990, 1991b,c) in: a seed, 83 µg; the 4-day dark grown sprout, 15 µg; the primary leaf, 15 µg; mature leaf, 100 mg; immature green flower bud, 35 µg; disk flower, 11 µg; cultured cells, 3,000 fg/cell; and the ray mesophyll cells released by macerase digestion, 500 fg/cell. Pollen grains were found to be the richest source of sterol, 585,000 fg/grain.
Sterols accumulate in vegetative parts mostly as 24-ethylsterols (e.g., sitosterol and stigmasterol [22-dehydrositosterol]) whereas pollen can accumulate significant amounts of 24-methylene sterols and 24-desalkylsterols (4,4-dinorcycloartenol; 24-dehydropollinastanol) (Guo et al., 1995; Nes and Schmidt, 1988). However, the sterols of some pollen and seeds can be mostly sitosterol and related phytosterols. Stem trichomes separated from sunflower had no detectable sterol within a GLC limit of detection set at <0.1% total sterol. Alternatively, the leaf wax contains cholesterol, as reported for sorghum leaves (Nes, 1990). Cycloartenol, usually an intermediate that accumulates to trace levels in the sterol mixture, can accumulate to as much as 25% in aging tomato leaves (unpublished data) and similarly can be a major sterol in soybean seeds (Marshall et al., 2001). The inability to detect cycloartenol in some instances may be due to the manner in which sterol was prepared for analysis. For instance, in many early studies sterol purification involved digitonin precipitation that fails to precipitate 4,4-dimethyl sterols along with the major phytosterols; therefore only the Δ5-sterol content in the tissues was determined.
The amount of total phytosterol in seedlings has been correlated to the level of SMT activity during plant maturation (Fig. 9.12). Transcript expression levels of SMT from Arabidopsis and soybean also vary during plant maturation
The phytosterol composition can change in regard to the structure of the sterol side chains with plant development. A ‘‘switching’’ mechanism in the biosynthesis of phytosterols has been shown in the cucurbits to produce different 24-alkyl sterol stereoisomers with development (Kalinowska et al., 1990). The seeds of squash and pumpkin synthesize 24β-ethyl sterols whereas the seedlings of
The principal approach adopted seeks to derive developmental and evolutionary understanding of sterol synthesis by combining the sterol and genetic composition of plants with sterol functions and the morphological evidence of plants at different time points. These studies indicate that sterol biosynthesis can proceed in plants by a cycloartenol pathway and in animals and fungi by a lanosterol pathway (Goodwin, 1981; Nes et al., 1990). In addition, the size and direction of the 24-alkyl group of the sterol side-chain can be an indicator of an early or late stage of development and whether the plants are less or more advanced. Primitive organisms synthesize phytosterols with a 24β-methyl group and vascular plants synthesize sterols with a 24α-ethyl group (Kalinowska et al., 1990). Since C-methylation is an energy expensive process, it seems unlikely that the cell would commit energy to produce the side-chains of ergosterol or sitosterol unless the methylation event was functionally significant. Although few pure enzymes of sterol catalysis have been examined in any detail, it does seem safe to suggest that a relatively small number of SMT enzymes determine the basic structural character of the phytosterol side chains produced in a given species.
The type and amount of phytosterol can change during the life history of plants and fungi yet there is a sterol homeostasis maintained at the cell level throughout development. The question arises as to why certain sterols accumulate and what biology maintains their balance in the cell. In other words, is one sterol as good as another? Can a single sterol play multiple roles? Is there something special about a particular sterol cocktail? One way to get at this problem would be to determine what sterols actually do, to find a way to quantify the value of individual features, and then to determine the extent to which deviations in sterol homeostasis affect function. The occurrence of intermediates is also problematic since different pathways can lead to the same end product. In the first case, the biosynthetic sequence determines the structure of the end product. In the second case, a choice presumably is made in a way that has no impact on the structure of the end product. Nes discussed the possible multiple roles of sterols in plants and fungi (Nes, 1980) and the difference between functional and phylogenetic control of biosynthesis (Parker and Nes, 1992). Evidence in support of phylogenetic control is the cycloartenol-lanosterol bifurcation whereby either precursor can give rise to ergosterol. In contrast, functional control is related to the structure and function of enzymes that act on sterols that, once impaired, interrupt flux to end products resulting in aberrant morphology or cell death. In support of the latter hypothesis is recent work from animal and plant studies that show mutations in the terminal segments of the cholesterol and sitosterol pathway lead to malformations (Clouse, 2002; Herman, 2003).
The hypothesis that sterols play multiple roles in plants and fungi has its origins with the work of Clark and Bloch (1959) who studied the insect nutritional requirements of sterol. Insects cannot make their own sterol due to a block in the pathway before squalene oxide cyclization, therefore they are dependent on an exogenous or dietary source of sterol to satisfy their structural and physiological needs for them (Clark and Bloch, 1959). Clark and Bloch discovered a sterol function other than the bulk membrane role for sterols on the basis of structural and quantitative requirements. Thus, feeding different amounts of a mixture of cholesterol and cholestanol to the insect resulted in the finding that cholestanol can spare (partly replace) cholesterol. The function of cholestanol was considered to be that of a regulatory molecule, not necessarily as a precursor to a hormone, for example, ecdysteroid. Fungi have been found to utilize sterols in multiple roles as well and various actions similar to sparing cholesterol in insects have been described with the yeast ergosterol (Nes, 1987b). The requirement for sparing amounts of a 24-ethyl sterol was demonstrated using cultured celery cells (Haughan et al., 1987). Support for the involvement of the different phytosterols in different physiological roles has been obtained with the fungus G. fujikuroi where there is delayed expression of certain sterolic enzymes that generate different sterol compositions (Nes and Heupel, 1986) and mutation studies in plant sterol synthesis that show changes in sterol composition affects morphology (Lindsey et al., 2003; Schaller, 2004).
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