Chemotaxonomy

Lecture 20

Chemotaxonomy

the attempt to classify organisms (originally plants), according to differences in their biochemical makeup

aka biochemical systematicsPlant biochemical characters have been used directly in taxonomy > 100 yrs Indirect use in classification through odours, colours, tastes & medicinal value

Started with herbalists who classified the plants according to their medicinal uses / chemical properties phytochemical characters attracted increased attention of biosystematists in recent decades (c. 1960s), as new & powerful analytical techniques have allowed the detection of very minute quantities of various chemicals.

Chemotaxonomy

Phytochemicals have been used to describe

Infraspecific variations Interspecific differences Phylogenetic relationships of families & higher-order taxa

More emphasis now placed on elucidating biochemical pathways rather than presence / absence of the phytochemical(s) themselves

Chemotaxonomy

Phytochemical characters correlate well with other types of characters and so derive their application to phytotaxonomy. Phytochemicals of taxonomic significance may be grouped as: 1. Primary metabolites 2. Secondary metabolites 3. Visible compounds

MiscellaneousSome 1 compounds like starch are also visible, as are some 2 ones, composing crystals

Chemotaxonomy - Primary compounds

These directly participate in the metabolism of the plant:

Carbohydrates Proteins Nucleic acids

Chlorophyll

Carbohydrates - visible

Starch:

develops as grains in plastids (amyloplasts). starch grains range from 1-100 m in diameter, according to their source, but in general are composed of successive lamellae lying parallel to the grain surface (whatever the local shape of the grain may be) lamellae arranged concentrically. therefore grain may differ in size, shape and arrangement of the concentric rings. used in the classification of the family Poaceae(aka Graminae).

Carbohydrates - visible

Starch in Poaceae:

Starch grains found in the endosperm of grasses display a remarkable degree of variation in size and organization.

The simple type of grain consists of a single granule whereas compound grains may be composed of 2-100+ individual granules. The nature of the grains has some value in grass systematics, especially at the subfamily level.

while simple grains are found in many taxa in the Panicoideae, taxa of the Festucoideae, Eragrostoideae and Chloridoideae are often characterized by compound grains.

Sorghum (Sorghum) endosperm contains many simple grains. (Panicoideae)

Wheat (Triticum) has simple starch grains that are widely variable in size. Small impressions on the large grain in the center were caused by pressure from other simple starch grains before the endosperm was prepared for scanning electron microscopy. (Festucoideae: note that many festucoids have compound grains)

SEM view of starch grains from wheat endosperm. Like other cereal endosperms, wheat has two clear-cut size categories, the smaller grains being referred to as -granules. (Pooideae)

Carbohydrate - visible

Fescue (Festuca) has compound grains composed of many, smaller angular granules. Note the two large compound grains in the center of the micrograph. (Festucoideae)

Carbohydrate - visible

Stinkgrass (Eragrostis) displays compound grains consisting of many angular granules. (Eragrostoideae)

Starch grains from the latex of Euphorbia species

The simplest (left) are round, but with growth become serrated at the periphery. Euphorbia latex contains many elongated, bone-shaped starch grains, which can develop new centres of deposition of starch and branch, then fill in the indentations with further deposition

starch grains viewed via differential interference contrast (DIC) optics (top row) and polarised light (lower row) showing symmetry patterns From L-R: wheat, potato, mung bean, Rhoeo

Crystals - visible

Occur in cytoplasm of ground tissue mostly

Chemically:

CaC2O4 (calcium oxalate) or Ca(HCO3)2 (calcium bicarbonate) or SiO2 (silica dioxide)Druses: spherical groups of crystals Raphides: needle-like crystals Prismatic bodies Silica bodies (phytoliths) Cystoliths: amorphous calcareous aggregates

Morphologically:

Druses

Druses

Raphides

Can be clearly seen in leaf sections and they occur in about 35 families of flowering plants. Within the Arecaceae:

Coryphoideae and Ceroxyloideae: embryos with raphides were rare, but within the Arecoideae: common feature of the tribes Areceae and Caryoteae.

Araceae: grooved raphide crystals

Dieffenbachia, Anthurium & Caladium

Rubiaceae: all the members which have raphides have been separated into a sub-family Ruboideae and this was also supported by other characters.

Proteins

Useful taxonomic information through:

Amino acid sequencing Serology Electrophoresis

Proteins - Amino acid sequencing

Useful phylogenetic information in the sequence of amino acids of proteins Reflective of nucleic acid patterns Proteins used to infer phylogeny of plant groups:

cytochrome c; plastocycyanin; ferredoxin; RuBP carboxylase (rbcL)

Recently the shift is toward the actual nucleotide sequencing of DNA. Promoted by the current technology available.

Proteins serology

The technique of serology relies on the immunological reactions shown by mammals when they are invaded by foreign proteins.

Proteins extracted from a particular plant species, usually from pollen or testa of seeds Injected into an animal such as a rabbit Antibodies produced These antibodies are then cross-reacted with antigens of taxa related to the original species These antibodies can be extracted from the blood of the animal as an antiserum. The degree of precipitation is indicative of the relatedness of the taxa

Proteins serology

If plant extract (A) results in the mammal producing proteinaceous antibodies, specific to an antigen and capable of coagulating it (and hence rendering it nonfunctional). Extract A will coagulate further supplies of antigens and it can be used as a standard test against other plant extracts (B, C, D, etc.) The degree of coagulation which it causes in them can be used as a measure of their similarity to plant extract A (and hence the similarity of species B, C, D, etc. to species A).

Proteins serology

Used to assess the phylogeny of:

Hydrastis, once placed in the in the Berberidaceae, is now proven to be more in the Ranunculaceae Nelumbo is now placed in the Nelumbaceae, once included in the Nymphaceae

Serologial comparison of major seed proteins showed a close relationship between the sub-classes Magnoliidae & Hamamelidae. Serological evidence has confirmed the transfer of Phaseolus aureus and P. mungo to the genus Vigna from the genus Phaseolus

Proteins electrophoresis

Measurement of the rate and direction of migration of a protein under the influence of an electric field

Pollen or seed coat proteins or specific enzymes(cytochrome; ferroxin etc.)

Starch or agar gel medium Dependent on the net surface charge, size & shape of the protein Protein staining leads to banding patterns on the gel Banding patterns compared

Presence / absence of particular bands analyzed Homologous proteins yield similar banding patterns thereby suggesting relatedness

Proteins electrophoresis

Chemotaxonomy - Secondary compounds

These secondary metabolites are also called secondary plant products

Once thought to have little or no role in the metabolism of the plant, now known to function in defense against predators (phytotoxins) and pathogens (phytoalexins) allelopathy: plant products which negatively influence the growth & development of surrounding plants attracting pollination and/or dispersal agents Insect hormones (coordinate growth of the insect with that of the plant) Simply end-products of metabolism (no apparent biological role or function)

Uses of plant secondary metabolites by humans

Drugs Medicines (cardiovascular, neurological, antitumor, antimicrobial) Narcotics Stimulants Hallucinogens Poisons (insecticides, rodenticides) Essential oils Resins for preservation Phenolics for tanning animal skins Dyes Rubber Chemotaxonomy

Chemotaxonomy - Secondary compounds

They include:

Alkaloids Phenolics Betalains & Anthocyanins Terpenoids

Main groups of 2 metabolites1.

Alkaloids a diverse group; are nitrogencontaining products mostly synthesized from amino acids.

Indole alkaloids Tropane alkaloid Isoquinoline alkaloids Isoprenoid alkaloids

Phenol

2.

Phenolics are a class of chemical compounds consisting of a hydroxyl group (-OH) attached to an aromatic hydrocarbon group. The simplest of the class is phenol (C6H5OH). Includes phenolic flavononids, tannins, and polyketides

Main groups of 2 metabolites

Volatile 2 metabolites3.

Terpenes

Aliphatic oils Aromatic (miscellaneous) odours Nitrogen containing volatiles (aminoid/fruity odors)

4. 5.

Sulphides Mustard oils (isothiocyanates, glucosinolates )

Alkaloids

Alkaloids are structurally diverse.

e.g. caffeine, nicotine, morphine, colchicine, quinine, cocaine, atropine, colchicine & strychnine Most alkaloids have a very bitter taste They are usually located

in vacuoles of alkaloid plants present in large amounts in storage organs. Sometimes they may also be present in large quantities in some leaves.

Ephedrine

Alkaloids

Alkaloids can be classified

in terms of their biological activity, chemical structure (nucleus containing nitrogen), biosynthetic pathway

Alkaloids can be of three main types based on their synthesis:

Derived from amino acids

True alkaloids: heterocyclic ring w/nitrogen Proto alkaloids: no heterocyclic ring w/nitrogen

Pseudo alkaloids: not derived heterocyclic ring w/nitrogen

from

amino

acids,

Alkaloids

Indole alkaloids characteristic of:

Apocynaceae, Loganiaceae, Gelsemiaceae & Rubiaceae of the Gentianales Solanaceae & Convolvulaceae of the Solanales Basal families in Magnoliales, Laurales, Ranunculales and in Nelumbonaceae

Tropane alkaloids characteristic of:

Isoquinoline alkaloids characteristic of:

Isoprenoid alkaloids show a more scattered distribution among angiosperms

Not very useful in taxonomical studies

Alkaloids

Morphine is an alkaloid which is only produced in Papaver somniferum (opium poppy) which belongs to Papaveraceae. The alkaloid protopine is common to all species of Papaveraceae. A species of another family Fumariaceae also contain protopine and this character has been used by taxonomists to show that these two families (Papaveraceae and Fumariaceae) are closely related.

Phenolics

Arguably the most taxonomically useful phytotaxonomic compound, partly due to ease of extraction.

These form a very loose class of compounds having in common only the fact that they are based upon phenol, C6H5OH. Most of them have no known functions in plants, but some are the most important flower pigments and others are involved in the inhibition of pathogenic fungi and anti-herbivory. Also present in leaves & fruits. These include flavonoids, leuco-anthocyanins etc. Useful in inferring phylogenies and naturally occurring interspecific hybridizations

Phenolics - flavonoids

Many positive correlations displayed by flavonoids Show an increase in complexity from the simple glycoflavones of Chara through to the proanthocyanidins of ferns, gymnosperms & primitive angiosperms

Phenolics - flavonoids

They are present in flower colour, pigment and act as insect attractants. Their mere presence/ absence is a useful character.

They have been used in understanding the generic relationships in the family Ulmaceae and species relationships in the genus Chenopodium. These flavonoids have also been useful in tracing the evolutionary pathway in the family Lemnaceae.

Phenolics - flavonoids

The leaf flavonoid chemistry in Liliaceae, Juncaceae, Poaceae and Cyperaceae supported the view that all of them have arisen form Liliaceous ancestors. The family Araceae was not thought to have been arisen from Liliaceous ancestors because Araceae contains different alkaloids which are not present in Liliaceae or other related families. Based on the presence of cyanidin 3, 5, 3 triglucoside in the sub-family Bromelioidae of the family Bromeliaceae, a chemical affinity was associated between this sub-family and the family Commelinaceae and sub family Commelinedae which have cyanidin 3,7,3 triglucoside.

Betalains & Anthocyanins

Betalains & anthocyanins are mutually exclusive Betalains Nitrogenous, red & yellow pigments The red colour of beets is from betalin, a glycoside Caryophyllaceae, Portulacaceae, Amaranthaceae Anthocyanins Purple, deep red pigments Found in most other plants

Betalains & Anthocyanins

Pollinator attractants Implicated in UV absorption & herbivory deterrence in young plants This dichotomy of their presence has assisted in the elucidation of the systematic position of several taxonomic groups

Dysphania was assigned at different times to either Caryophyllaceae or Chenopodiaceae by various authors, but now is strongly placed in Chenopodiaceae based on the presence of Betalains Giseka once in Molluginaceae, now assigned to Phytolaccaceae; again due to presence of Betalains

Glucosinolates

Glucosinolates

aka. Mustard oil glycosides

Hydrolysed to give pungent, hot mustard oils Brassicaceae; Moringaceae; Tovariaceae; Resedaceae

Synapomorphic for the Brassicales

Cyanogenic Compounds

Hydrolyzed to release hydrogen cyanide (cyanogenesis) when injured occurring in:

Rosaceae

FabaceaeSapindaceae

The comparison of cyanogenic glycosides in Liliopsida and extant Magnoliidae, suggest that the former was derived ancestors of the latter. Biogenesis of cyanogenic compounds are characteristic of certain taxa based on their foundation compounds:

Pteridophytes phenylalanine only

Gymnosperms tyrosine onlyAngiosperms valine, leucine, glycine, in addition to phenylalanine & tyrosine

Terpenoid compounds

Large & structurally diverse group Widely distributed

Membrane steroids Carotenoid pigments Phytol chain of chlorophyll Hormones: GA & ABA

Terpenoid compounds

There are different groups of terpenes:

Monoterpenes (10-C)Diterpenes (20-C) triterpenes (30-C)

sesquiterpenes (15-C)

Terpenoids were used in identifying 3 sympatric (i.e. common origin and parallel development) taxa of the genus Hedeoma (Lamiaceae) that coexisted w/o any sign of natural hybridization; using their terpenoids to distinguish them.

Terpenoid compounds

Volatile monoterpenoids & sesquiterpenoids are major components of essential (ethereal) oils in Magnoliales, Piperales & Laurales as well as the eudicots: Myrtaceae, Rutaceae, Lamiaceae, Verbenaceae & Asteraceae Cymbopogon (lemon grass) & Vetiveria (khus khus) owe their distinctive odour to terpenoids The triterpenoid betulin is unique to the Betulaceae and is taxonomically useful even at the species level The presence of triterpenoid saponins in Apiaceae & Pittosporaceae indicate their close phylogenetic relationship The related quassinoids of the Simaroubaceae (Red birch group) are distinctive to the group