With the enormous advances made in the field of phytochemistry, a vast number of compounds have been isolated and characterized. Some of them have been recognized as being chemotaxonomically important.
The phytoconstituents that are useful in chemotaxonomy are classified as: 1. Primary Metabolites 2. Secondary Metabolites 3. Semantides and Macromolecules.
1. Primary Metabolites:
These are the products of vital metabolic pathways i.e., products of the respiratory chain, TCA cycle, fatty acids and essential amino acids etc. They are of universal occurrence, and thus, lack systemic significance in chemotaxonomy. However, in certain organs of a few plants, they are stored in excess. Apart from their accumulation, the nature of accumulation is important.
For Example:
i. Sac-shaped starch grains in ginger and turmeric
ii. Sucrose, though of widespread occurrence, is present in large quantities in sugarcane and beetroot.
Quantitative variations often serve as a ‘chemotaxonomic substance’. For instance, the presence of relatively high amounts of the amino acid, arginine, in the family Rosaceae, irrespective of its common occurrence in the plant kingdom, is chemotaxonomically significant.
2. Secondary Metabolites:
Secondary metabolites are less widespread in the plant kingdom, as they perform only non- vital functions. They are usually large molecules with many side chains. The secondary metabolites that have received attention in chemotaxonomy are alkaloids, glycosides, terpenoids, volatile oils, flavonoids etc. Their role in plant classification has been increasingly recognized.
Biosynthetic Complexity and the Occurrence of Secondary Metabolites:
The relationship between the biosynthetic complexity of a secondary metabolite and its taxonomic significance is discussed here.
The occurrence and distribution of a phyto-constituent in the plant kingdom depends on its biosynthetic complexity. The more difficult the reaction for the formation, the less likely it is to occur independently in unrelated plants. Therefore, the biosynthetic complexity of a compound determines its occurrence i.e., whether it occurs only in a limited number of species, or in an entire family or order of the plant etc.
For example, the biosynthetically complex alkaloid, morphine, is found only in two species of Genus Papaver (P. somniferum and P. setigerum). On the other hand, the relatively simpler alkaloid protopine, occurs in all plants of the poppy family.
Thus, those compounds, which are biosynthetically simple, are widely distributed, hence, lack systemic significance. Similarly, those compounds which are present in only a few species are also of little interest, unless biosynthetically similar compounds occur in other plants.
Chemical compounds, though diverse in their nature, occurring in different plants, appear to be biosynthetically analogous. Such plants probably contain similar enzyme systems in them for their biosynthesis. The formation of these compounds reflects a relationship (for example, having similar enzyme systems) between them.
Apart from formation, their accumulation is another important aspect. Some secondary metabolites like nicotine and related alkaloids are produced in trace quantities by many plants, but accumulated in large quantities only in few genera such as Nicotoiana and Duboisia.
Secondary Metabolites in the Perspective of Chemotaxonomy:
Phenolic Compounds:
Phenoloic compounds, so far, have provided the maximum taxonomic data for chemotaxonomy. Among them, flavonoids have been extensively studied. They have a common nucleus, with a great variety of types and patterns of side chains.
Two classes of water-soluble pigments – anthocyanins and betacyanins – are responsible for the red, purple and blue colours in many plants. The anthocyanins are flavonoids, whereas betalains (including the betacyanins) are nitrogen-containing heterocycles, while performing the same functions as the former. The anthocyanins are biosynthesized via the shikimic acid pathway, while betalains are formed by an altogether different metabolic route.
The distribution of anthocyanins and betalains in the plant kingdom is chemotaxonomically important. The occurrence of anthocyanins is widespread in the plant kingdom, ranging from mosses to gymnosperms, as well as in the angiosperms. These are absent in a few families, which alternatively contain betalains – this is an interesting point as it reveals the presence of one and the absence of the other metabolic pathway.
Betalains as Chemotaxonomic Markers:
A total of 9 families contain betalains, out of which 7 are placed in the order ‘Centrospermae’. The other 2 families containing betalain come under the orders ‘Cactales’ (Cactaceae) and ‘Sapindales’ (Didicraceae). The other families namely, Gyrostemonaceae, Caryophyllaceae and Mollginaceae, of the order ‘Centrospermae’, are free from betalains, but contain anthocyanins.
Hence, from the perspective of chemotaxonomy, the inclusion of these 9 families containing betalains in ‘Centrospermae’, and excluding the remaining 3 families from it, is ideal. Similarly, the species of the genera ‘Primula’ can be distinguished on the basis of its petal flavonoids.
Alkaloids:
Alkaloids are nitrogen-containing bases, usually with a heterocyclic ring. Though they generally lack significance in chemotaxonomy, alkaloids assume importance in a few cases due to their specific distribution.
True alkaloids are of rare occurrence in the lower plants, such as ephedrine in ephedra (a gymnosperm), annotine type of alkaloids and traces of nicotine in lycopodium (a pteridophyte). The distribution of alkaloids in angiosperms is uneven. Certain groups of alkaloids have been associated with particular families or genera.
Some of the significant chemotaxonomical findings are discussed below:
i. The inclusion of families Solanaceae and Convolvulaceae in the same order is justified as they contain tropane alkaloids.
ii. Similarly due to the presence of benzyl isoquinoline and the absence of glucosinolates, it may be concluded that the Papaveraceae family is nearer to Ranunculaceae than Cruciferae and Capparaceae.
iii. Papaveraceae and Fumariaceae are closely related, as both contain the alkaloid protopine.
Glycosides:
Glycosides, occurring in plants and animals, are acetals chemically, which on hydrolysis yield one or more sugars along with a non-sugar moiety. They are classified on the basis of the linkage between the sugar and non-sugar moiety.
The distribution of the various types of glycosides is discussed below:
‘O’-Glycosides:
Among glycosides, ‘O’-glycosides are widespread, and are therefore, less important in terms of chemotaxonomy. But, in a few cases, they may attain some importance. For example, the nature of sugar moiety, like de-oxy sugars in cardiac glycosides or the unusual position of attachment of sugars to aglycone may be characteristic.
‘C’-Glycosides:
In these glycosides, sugars are attached to the aglycone by carbon-carbon linkage. These are relatively fewer in occurrence. Aloin (aloes), cascarosides (cascara bark) are important anthraquinone derivatives which are ‘C’-glycosides.
About 15 ‘C’ glycosides of the flavonoid group are present in 8 unrelated families, ranging from ferns to monocotyledons, but in greater amounts in Papilionaceae.
‘S’-Glycosides:
These glycosides give isothiocyanate on hydrolysis. The occurrence of thioglycosides in all species of the families of Cruciferae, Capparaceae, Papaveraceae and Fumariaceae unites them into a natural group, under the order ‘Rhoedales’.
Based on chemical and other evidence, Cruciferae and Capparaceae are now included in the order Capparales (containing glucosinolates), whereas Papaveraceae and Fumariaceae are placed in the order ‘Papaverales’ (not containing them). Some ‘S’-glycosides occur in 8 other unrelated families, but are not characteristic of them, as they are present sporadically.
Cyanogenetic Compounds:
These are distributed in about 80 families of angiosperms, a few gymnosperms and several fungi. They hold some systemic significance in chemotaxonomy because of their limited distribution and also ease of their detection. In fact, they are extensively studied for this purpose.
The presence of the same biogenetic type of cyanogenetic glycosides in closely related plant species establishes the familial relationship between them. For example, only phenyl alanine-derived glycosides, such as prunasin and vicianin, are present in fern, while only the tyrosine-derived taxiphyllin is found in gymnosperms.
The original order, Perietales, was taxonomically divided into two orders – Guttiferales and Violales. This division appears to be in accordance with chemotaxonomic principles. A number of families belonging to the sub-order Flacoutinae of Violales are rich in cyanogenetic glycosides; but no genera of the family Violaceae, whose inclusion in this order is doubtful, has them. Sixteen families of Guttiferales also do not contain them.
The family Rosaceae is known for the presence of cyanogenetic glycosides. Earlier, it was believed that the presence of phenyl alanine-derived glycosides, such as prunasin and amygdalin, is characteristic of this family. Later, it was proved true only in the case of two subfamilies, as the other subfamily was reported to contain heterodendrin, and cardeospermin-p-hydroxybenzoate, cardeospermin-p-hydroxycinnamate or dhurrin. Therefore, the division of this family into subfamilies is in accordance with the concept of chemotaxonomy using cyanogenetic glycosides as the taxonomical marker.
Terpenoids:
These are a varied group of plant constituents formed by the condensation of active isoprene units. Irrespective of their complex structures, some of them like phytol, carotene and sterols are found in all phyla of the plant kingdom as they are physiologically vital. They therefore lack systemic significance in chemotaxonomy.
Carotenoids are associated with all photosynthetic tissues, and therefore, occur with chlorophyll in higher plants. The con-elation between the natures of the carotenoid content in various algae of the proposed evolutionary development, which is based on conventional assessment, is presented in Table 14.1.
Table 14.1 Relationship between the Nature of Carotenioid Content and Different Classes of Agae:
In higher plants, the conversion of chloroplasts into chromoplasts occurs during ripening with the formation of products like β-carotene oxidated products. These are devoid of any significance, except a in a few, like capsanthin (capsicum).
The extensive work that was carried out worldwide revealed the link between the formation of triterpenes and phylogeny. The details are given in Table 14.2.
Table 14.2 Relationship between the Nature of Terpenes and Status of Plants in Plant Evolution:
Many important sesqui-terpene lactones are reported in the family Compositae, and their nature and distribution have been studied in detail. These studies revealed that the subdivisions of this family are characterized by the distinct types of the sesqui-terpene lactones they biosynthesize.
Irridoids:
These are another important group of terpenes, mostly monoterpene lactones. Their presence in 50 families is correlated with certain of their morphological features. Therefore, all these can be brought into the same group chemotaxonomically. However, certain unrelated families also contain them, which indicate that their formation in them is probably independent of the evolutionary process of angiosperms.
3. Semantides and Macromolecules:
Semantides are the genetic information-carrying molecules.
They are classified as:
i. Primary semantide – DNA.
ii. Secondary semantide – RNA.
iii. Tertiary semantide – Proteins.
Owing to their large molecular size as well as complex structure, the isolation and comparative study of semantides is very difficult. In view of this, special techniques like DNA-DNA hybridization, serology, electrophoresis, amino acid sequencing etc., are required for gaining relevant evidence in the case of semantides.
The sequence of nucleosides, in case of nucleic acids, provides the necessary information for chemotaxonomy.
DNA-DNA Hybridization:
Charles Sibley and John Ahlquist were the pioneers of this technique, which is still widely used in microbiology to identify bacteria. This molecular biology technique is a fundamental technique available in comparative phytochemistry; it involves the comparison of DNA molecules from different species.
In this technique, the degree of genetic similarity is determined to find out the genetic distance between two species. DNA sequencing and computational comparisons of sequences is now the generally adopted method for determining genetic distance.
DNA exists as a double helix and its two complementary strands (helices) can be separated (dissociated) from each other by heating to 100˚C and cooling rapidly. Under suitable conditions, they come together again and combine to form a complete molecule (re- associated DNA). Re-association depends on the collision rate i.e., concentration.
In the procedure, DNA from the two species to be compared is extracted, purified and cut into short pieces of 600-800 base pairs. The DNA of one species is labelled, and then mixed with the unlabelled DNA of the second one. The mixture is incubated to allow the DNA strands to dissociate and re-anneal, forming hybrid double-stranded DNA.
The technique of DNA melting’ is utilized for comparing one species with another. ‘DNA Melting’ is a measure of the energy or temperature required for the separation of the strands of DNA. It represents the thermal stability of re-associated DNA molecule. The degree of base pair matching along the double strands of the re-associated DNA molecule determines the thermal stability.
Hybridized sequences with a high degree of similarity will bind more firmly, and therefore, require more energy to separate them. Therefore, native DNA will have higher stability because of perfect matching than the hybridized DNAs which have some degree of base pair mismatching.
In this way, species, varieties etc., of plants can be compared on the basis of the extent of their DNA Hybridization.
Proteins:
These are probably only next to phenolic compounds in providing the data required for chemotaxonomy. Proteins, though widely occurring in plants and animals, still possess some chemotaxonomical importance, as they differ from species to species. The comparison of a protein mixture provides the basis for the possible chemotaxonomic grouping of such species.
The methods employed for obtaining the chemotaxonomic data are:
i. Serology.
ii. Electrophoresis.
iii. Amino acid sequence.
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