AUXINS

These growth-promoting substances were first studied in 1931 by Dutch workers who isolated two growth-regulating acids (auxin-a and auxin-b, obtained from human urine and cereal products, respectively). They subsequently noted that these had similar properties to indole-3-acetic acid (IAA), the compound now considered to be the major auxin of plants, and found particularly in actively growing tissues. Several similar acids, potential precursors of indoleacetic acid, have also been reported as natural products; they include indoleacetaldehyde, indoleacetonitrile and indolepyruvic acid. These compounds and IAA are all derived, in the plants, from tryptophan.

Typical effects of auxins are cell elongation giving an increase in stem length, inhibition of root growth, adventitious root production and fruit-setting in the absence of pollination. A number of widely used synthetic auxins include indole-3-butyric acid, naphthalene-1-acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D).

In the plant, oxidative degradation of IAA to give a number of products is controlled by IAA oxidase. Some substances such as the orthodiphenols (e.g. caffeic and chlorogenic acids and quercetin) inhibit the action of the enzyme and, hence, stimulate growth themselves. Conversely, monophenols such as p-coumaric acid promote the action of IAA oxidase and so inhibit growth. IAA may also be conjugated in the plant with aspartic acid, glutamic acid, glycine, sugars and cyclitols; such bound forms may represent a detoxication mechanism or are inactive storage forms of the hormone.

The main practical uses of auxins are: (1) in low concentrations to accelerate the rooting of woody and herbaceous cuttings; and (2) in higher concentrations to act as selective herbicides or weed-killers. Placed for 24 h in a 1:500 000 solution of NAA, cuttings will subsequently develop roots. This includes cuttings from trees such as holly, which were formerly very difficult to propagate in this way and had to be raised from seed or by grafting. Similarly, indole-3-butyric acid was successful with Cinchona cuttings, saving some 2 or 3 years compared with growth from seed. Similar results have been obtained with cuttings of Carica, Coffea, Pinus and other species. In biogenetic studies, use has been made of auxins to induce root formation on isolated leaves such as those of Nicotiana and Datura species. Auxins used in suitable concentration (usually stronger than when used for rooting cuttings) selectively destroy some species of plant and leave others more or less unaffected. They have, therefore, a very important role as selective weed-killers in horticulture and agriculture. Thus 2,4-D is particularly toxic to dicotyledonous plants while, in suitable concentration, having little effect on monocotyledons. It can, therefore, be used to destroy such dicotyledonous weeds as dandelion and plantain from grass lawns. (N.B. Certain carbamate and urea derivatives have an opposite effect and can be used to destroy grass without serious injury to dicotyledonous crops.)

There have been several reports on the effects of auxins on the formation of secondary metabolites on medicinal plants.

Seedlings and young plants of Mentha piperita, when treated with derivatives of NAA, gave in the mature plants an increased yield (30–50%) of oil which itself contained 4.5–9.0% more menthol than the controls. The study of the effects of auxins on alkaloid formation has concentrated principally on the tropane alkaloids of Datura species. Morphological changes in the plants were observed (2,4-D, for example, produced abnormal and bizarre forms of D. stramonium; an increase in trichome production, particularly in branched non-glandular forms; smooth fruits as distinct from those with spines; and a proliferation of vascular tissue). Generally, workers found no marked effect on alkaloid production or on the type of alkaloids produced, although a Russian paper records that with thornapple and scopolia tissue cultures a stimulating effect on alkaloid production was obtained with NAA and an inhibiting effect with 2,4-D; similar results were reported for Rauwolfia serpentina tissue cultures with these two hormones. An increased alkaloid production has been reported for submerged cultures of certain ergot strains when treated with various auxins (IAA; NAA; 2,4-D; indole propionic acid; indole butyric acid), whereas unpredictable irregular quantitative and qualitative effects on ergoline alkaloid production were observed with the same hormones in Ipomoea, Rivea and Argyreia (Convolvulaceae) suspension cultures. Experiments carried out in Hungary involving the injection of IAA into poppy capsules 1 and 2 days after flowering produced a relatively elongated capsule form and, in general, a reduced alkaloid content. In studies on anthraquinone production by cell suspension cultures of Morinda citrifolia, Zenk and co-workers have shown that cells grown in the presence of NAA have a substantial anthraquinone production but those with 2,4-D as sole auxin do not. IAA appears to have no beneficial effect on the production of sennosides in Cassia angustifolia.

GIBBERELLINS

This group of plant growth regulators was discovered by Japanese workers in connection with the ‘bakanae’ (foolish seedlings) disease of rice. In this, the affected plants become excessively tall and are unable to support themselves; through a combination of the resulting weakness and parasite damage they eventually die. The causative organism of the disease is Gibberella fugikuroi, and in 1926 Kurosawa found that extracts of the fungus could initiate the disease symptoms when applied to healthy rice plants. Some 10 years later, Yabuta and Hayashi isolated a crystalline sample of the active material which they called ‘gibberellin’. Preoccupation with the auxins by western plant physiologists, the existence of language barriers and the advent of World War II meant that a further 10 years elapsed before the significance of these findings was appreciated outside Japan. In the 1950s groups in Britain, the USA and Japan further investigated these compounds, which were shown to have amazing effects when applied to plants. It soon became apparent that a range of gibberellins was involved, and they are now distinguished as GA₁, GA₂, GA₃, etc. GA₃, commonly referred to as gibberellic acid and produced commercially by fungal cultivation, is probably the best-known of the series; its structure was finally determined in 1959. The first good indications that gibberellins actually existed in higher plants came with West and Phinney’s observation in 1956 that the liquid endosperm of the wild cucumber (Echinocytis macrocarpa) was particularly rich in substances possessing gibberellin- like activity and Radley’s report of a substance from pea-shoots behaving like gibberellic acid on paper chromatograms. Finally, in 1958 MacMillan and Suter isolated crystalline GA₁ from Phaseolus multiflorus. By 1980, 58 gibberellins were known of which about half were derived from the Gibberella fungus and half from higher plants. GA₁₁₇ was characterized from fern gametophytes in 1998 (G. Wynne et al., Phytochemistry, 1998, 49, 1837). Of these many GAs most are either dead-end metabolites or are intermediates in the formation of active compounds; only a limited number have hormonal activity per se. It is now considered possible that these substances are present in most, if not all, plants.

Gibberellins are synthesized in leaves and they accumulate in relatively large quantities in the immature seeds and fruits of some plants. The most dramatic effect of gibberellins can be seen by their application to short-node plants—for example, those plants producing rosettes of leaves (Digitalis, Hyoscyamus)—when bolting and flowering is induced; also, dwarf varieties of many plants, when treated with the hormone, grow to the same height as taller varieties. Other important actions of the gibberellins are the initiation of the synthesis of various hydrolytic and proteolytic enzymes upon which seed germination and seedling establishment depend.

The growth effect of gibberellins arises by cell elongation in the subapical meristem region where young internodes are developing. The effects of gibberellins and auxins appear complementary, the full stimulation of elongation by either hormone necessitating an adequate presence of the other.

As with auxins, gibberellins appear also to occur in plants in deactivated forms; thus, β-D-glucopyranosyl esters of GA₁, GA₄, GA₈, GA₃₇ and GA₃₈ are known. As such they may serve a depot function. The glucosyl ester of GA₃ has been prepared in several laboratories.

The biogenetic pathways of the gibberellins appear to be similar in both higher plants and Gibberella. They arise at the C₂₀ geranylgeranyl pyrophosphate level of the isoprenoid mevalonic acid pathway (q.v.) with cyclizations giving the C₂₀ tetracyclic diterpernoid entkaurenoic acid, which by a multistep ring contraction furnishes the gibbane ring system as exemplified by the key intermediate GA₁₂-aldehyde. Several pathways diverge from GA₁₂-aldehyde to give the known 90 or so gibberellins. All GAs have either the ent-gibberellane (C₂₀ GAs) or the ent-20-norgibberellane (C₁₉ GAs) (loss of C-20) carbon skeleton. Both types are modified by the position and number of OH groups, oxidation state of C-18 and C-20, lactone formation, presence and position of double bonds on ring A, epoxide formation, the presence of a carboxyl group and hydration of the C16–C17 double bond.

The gibberellins have been used to treat many plants which contain useful secondary metabolites. A summary of some of the findings, for different groups, is given below.

CELL DIVISION HORMONES: CYTOKININS

Auxins and gibberellins are concerned largely with cell enlargement; and although they influence cell-multiplication processes, there are other substances which have a more specific effect on cell division (cytokinesis). The activity of the latter is not only confined to cell division in a tissue per se; they also regulate the pattern and frequency of organ production as well as position and shape. They have an inhibitory effect on senescence. The presence of such compounds has been suspected for many years, the German botanist Haberlandt in 1913 having noted that phloem tissues contained water-soluble substances capable of promoting cell division in parenchymatous cells of wounded potato tubers. It was not until many years later (1954) that Miller, working at Wisconsin on tissue cultures, discovered that aged or autoclaved DNA from herring sperm stimulated cell division. This active degradation product was called kinetin and in 1955 was identified as 6-furfurylaminopurine (6-furfuryladenine). Kinetin itself has not been isolated from plants, but in 1964, after the indication of cytokinins in liquid endosperm of the coconut and in extracts of maize embryos at the milky stage, an active substance named zeatin was isolated from the latter source. Like most other cytokinins, it is a 6-substituted adenine derivative, 6-(4-hydroxy-3-methylbut-2-eny1)-aminopurine. It has since been shown to be associated in maize with zeatin riboside (1 β-D-ribofuranose) and with a phosphate ester of this compound. The hormone complex has been detected in the cambial region of various woody plants. Isopentenyladenine and dihydrozeatin are examples of cytokinin isolated from other sources; many more have been detected but not identified. The sidechain of cytokinins is of isoprenoid origin.

Cytokinins have been much employed in tissue culture work, in which they are used to promote the formation of adventitious buds and shoots from undifferentiated cells. In cell cultures, they have been shown to promote the biosynthesis of berberine (Thalictrum minus), condensed tannins (Onobrychis viccifolia) and rhodoxanthin (Ricinus).

A limited study only has been made of the effects of cytokinins on secondary metabolism in intact plants. Concerning plants producing tropane alkaloids, Ambrose and Sciuchetti have compared the action of kinetin and GA on Datura meteloides. Plants received weekly doses of 25 μg kinetin; in relation to the controls the following differences were noted:

Kinetin treatment	Gibberellic acid treatment
Shorter and bushier plants	Taller and spindly plants
Decreased growth	Increased growth
No change in alkaloid content in plant organs	Decreased alkaloid production
Delayed response	Rapid response

Luanratana and Griffin (J. Nat. Prod., 1980, 43, 546, 552; 1982, 45, 270) observed the beneficial effects of a commercial seaweed extract containing cytokinin activity on Duboisia hybrids grown both hydroponically and in a commercial plantation. In the latter there was an 18% increase in leaf yield and a 16% increase in hyoscine content compared with the controls. A further pointer to the usefulness of the treatment was that in field plants it led to a delay in the usual seasonal fall in alkaloid content (February–April in Australia), thus permitting an economically useful, extended collection period. Shah et al. (1990) reported favourable increases in growth and alkaloid yield with Hyoscyamus muticus treated with kinetin (50 p.p.m.).

Verzár-Petri injected benzyladenine and kinetin separately into developing poppy capsules; the effects were similar to those observed with auxins. Leaves of the coffee plant after kinetin treatment developed a transient increase of up to 10% in their caffeine content. The effect was transitory and passed after 6–12 days. With Cassia angustifolia plants low concentrations of hormone were found to increase slightly the sennoside content and to favour an increase in the dry weight of shoots.

GROWTH INHIBITORS

Natural growth inhibitors are present in plants and affect bud opening, seed germination and development of dormancy. One such substance, abscisic acid [3-methyl-5-(1-hydroxy-4-oxo-2,6,6-trimethyl-2-cyclohexane-1-y1)-cis,trans-2,4-pentadienoic acid] was isolated and characterized in 1965; it has also been isolated from the fungus Cenospora rosicola.

The structural similarity of abscisic acid (ABA) to the carotenoids prompted research on the relationship of these two groups of compounds and it has now been demonstrated that some xanthophylls, particularly violaxanthin, produce a germination inhibitor on exposure to light. Evidence has now accumulated in support of an indirect ‘apo-carotenoid’ pathway for ABA biosynthesis, the most likely precleavage precursors being 9′-cis-neoxanthin and 9-cis-violaxanthin which fracture across the 11,12 (11′,12′) double bond to produce xanthoxin which in plant tissues is readily transformed into ABA (see A. D. Parry and R. Horgan, Phytochem., 1991, 30, 815). However, it is also possible that ABA arises from farnesol at the C₁₅ sesquiterpenoid level of the MVA pathway (q.v.). The theoretical postulation involves a number of steps in which, as with the apo-carotenoid pathway, cis-Δ²′-xanthoxin could also be involved. In accord with isoprenoid biosynthesis, mevalonic acid (MVA) has been demonstrated to be stereochemically incorporated into ABA in higher plants, and likewise labelled acetate in Cenospora rosicola. Other substances related to abscisic acid havebeen isolated from plants and include vomifoliol (several sources), which lacks the 2,4-pentadiene side-chain; it has the same activity as abscisic acid in stomatal closure tests. Little or no work appears to have been reported on the effects of abscisic acid on the production of pharmacognostically interesting substances.

A number of synthetic growth inhibitors have been studied; the first to be described was maleic hydrazide in 1949. N-Dimethyl-aminosuccinamic acid can be considered as a hydrazine derivative and acts as a shoot-elongation inhibitor by suppressing the oxidation of tryptamine to IAA. Sciuchetti and colleagues showed that this compound sprayed on to Datura stramonium and D. innoxia plants reduced the eventual height of the plants and lowered, overall, their total alkaloid content; however, significant increases were noted in the concentrations of stem alkaloid (56% increase in the second week and 90% in the fourth week compared with the controls). The inhibitor, tributyl 2,4-dichlorobenzylphosphonium chloride (phosphon) produced similar results with D. ferox. Trigonelline, an alkaloid of fenugreek seeds, promotes cell arrest in G₂ (a specific period preceding mitotic division of the nucleus) in various legumes.

ETHYLENE

It has been known for many years that ethylene induces growth responses in plants, and in 1932 it was demonstrated that the ethylene evolved by stored apples inhibited the growth of potato shoots enclosed with them; it has a role in fruit ripening. Current thought maintains that this simple compound should be included among the natural plant hormones. Ethylene is synthesized in the plant from S-adenosylmethionine via the intermediate 1-aminocyclopropane-1-carboxylic acid (ACC). The gene for ACC synthase has been cloned from tomato squash (H. Klee and M. Estelle in Annu. Rev. Plant Physiol., 1991, 42, 529). One biochemical action of ethylene is the stimulation of the de novo synthesis and secretion of cell-wall dissolving enzymes such as cellulase during leaf abscission and fruit ripening. A compound that gives rise to a typical ethylene response in plants is (2-chloroethyl)phosphonic acid (ethephon) applied in aqueous solution in concentrations of the order of 100–5000 p.p.m. In the cell sap, at pH values above 4.0 it is broken down to ethylene and phosphate. It is marketed as Ethrel.

At low concentration ethylene has been shown to increase the sennoside concentration in Cassia angustifolia, and applied to tobacco leaves it stimulates production of the stress compounds phytuberin and phytuberol (these are compounds produced in response to tobacco mosaic virus); with Digitalis lanata tissue cultures, cardenolide accumulation is decreased. Ethephon is now increasingly used as standard practice for enhancing the flow of rubber latex. Sprayed on to the scraped bark (tapping groove) of the rubber tree it increases latex yields by from 36 to 130%.