In this article we will discuss about Indole-3-Acetic Acid (IAA) and Auxin in Plants.
Natural and Synthetic Auxin:
The main auxin in plants is indole-3-acetic acid (IAA Fig. 20-2). Additionally, several other naturraly occurring indole derivatives are reported to express auxin activity, including indole-3- ethanol, indole-3-acetaldehyde, and indole-3-acetonitrile.
It may be noted that all these compounds are precursors of IAA and their activity is in all probability due to conversion to IAA in the tissue. Recently, indole butyric acid (IBA) (Fig. 20-2) has been isolated from seeds and leaves of maize and potato tuber peel.
A chlorinated analog of IAA (4-chloroindoleacetic acid, or 4-chloro IAA; II Fig. 20-2) has also been isolated from the seeds of some legumes. Further, phenylacetic acid (PAA; Fig. 20-2) has been reported to have auxin activity. For details a reference may be made to a recent publication by Malik (1999).
All the four auxins are structurally similar and elicit several responses as IAA, and hence it is strongly suggested that they are all naturally occurring auxins. It remains to be ascertained whether the latter three auxins are converted to IAA in the tissue. It may be added that all the four natural auxins have an acidic side chain on an aromatic ring.
Biosynthesis of IAA: Pathways of Biosynthesis, Multiplicity of IAA Sources:
IAA is known to be synthesized through several pathways which include shikimate which gives rise to indole ring de nove. Most common route is conversion of tryptophan to IAA which is regarded as IAA synthesis. IAA formation has also been suggested through hydrolysis of IAA conjugates. In some instances IAA is transported from one region to another and is available as active IAA.
Tryptophan as a Precursor:
In the following figure pathways or biochemical reaction leading to the synthesis of indole acetic acid (IAA) and its derivatives are shown (Fig. 20-2A).
Went (1928) provided experimental evidence for indole acetic acid from the tips of Avena coleoptile. This is now widely used as a curvature test or Went technique. Coleoptile tips (Fig. 20-3) are cut and placed on small agar blocks. After a short while the agar block is cut into small pieces and applied to one side of coleoptile. It is observed that coleoptiles bend towards the opposite side.
Apparently some hormone has diffused from the coleoptile into the agar block (Fig. 20-2). When this agar piece is placed on the side of Avena tip the hormone diffuses down and triggers the intensified longitudinal growth in the Avena tissue.
This hormone is identified as indole acetic acid (IAA) and is also called growth substance or auxin. Indole acetic acid exists in plants either in the free or bound state. It may be esterified to gulcose or bound in peptide linkage with some amino acids, e.g., glutamic acid or aspartic acid.
The Tryptamrne Pathway:
Though limited in occurrence, tryptamine is also shown to be converted to indole-3-acetaldehyde.
The Indole-3-ethanol Pathway:
Indole-3-ethanol in probability is product of side reaction and is identified as a naturally-occurring component.
Of the different routes, the biosynthesis of IAA from tryptophan is a well-accepted route. However, the control point lies some-where between tryptophan and IAA.
In some species e.g., Brassicaceae IAA is shown to be formed from indole-3-acetonitrile (IAN), an indole derivative. The latter applied exogenously shows auxin activity probably by conversion to IAA through action of a nitrilase enzyme.
In Arabidopsis several tryptophan-requiring mutants have been isolated and two mutants contain elevated levels of IAN and IAA and theses are trp 2 and trp 3 mutants. This evidence points towards the distance of tryptophan-independent pathway for auxin biosynthesis in Arabidopsis. Possibly IAN is derived from glucobrassicin.
Hydrolysis of IAA Conjugates to Provide IAA:
Both seeds and vegetative tissue abound in amide linked and ester conjugates of IAA. Sometimes conjugates comprise as much as up to 90% of the IAA. In tissue cultures IAA conjugates provide slow and gradual form of IAA release. In general it is assumed that conjugates provide a major source of IAA for seedlings.
Using 14C-labelled tryptophan, direct evidence has been provided for the biosynthesis of IAA from tryptamine especially in meristem of young parts of the plant, e.g. shoot apex or embryos.
The polar translocation of auxin is responsible for its specific action. It is transported in a polar fashion (Fig. 20-4) and moves slowly. However, several, recent experiments with radioactive (IAA) have shown its acropetal swift movement.
It is usually assumed that auxin may move in opposite directions simultaneously. A specific correlation between auxin transport and growth regulating mechanisms has also been established.
Auxins are continuously produced in tissues of plants but do not accumulate in large quantities. A built in destroying process or inactivation of auxin in the system is also visualized. It may be degraded by several bacteria or may be irreversibly oxidized to a compound called methyleneoxindole by an enzymatic reaction. This compound may act as a growth inhibitor (Fig.20.5).
IAA can also be rapidly inactivated by its conjugation with several chemicals including aspartic acid (forming aspartyl-IAA) or several other amino acids, some sugars and polysaccharides and even proteins. Some of these conjugates are only temporarily inactive. This is one of the methods to reduce IAA concentration in a tissue. IAA is also supposedly translocated in the form of a conjugate.
Several factors, e.g., blue light, UV, riboflavin, and some enzymes (peroxidase, IAA-oxidase) degrade IAA. For their active action they require monophenols as cofactors. It is generally suggested that flavonol regulates the activity of IAA through its effect on IAA oxidase.
Methods of Determination:
In recent years sensitive techniques like gas chromatography, colorimetric and fluorometric assays and mass spectrometry GC-MS are used to measure auxin levels. A new method for auxins extraction has been proposed which prevents simultaneous extraction of indole pyruvic acid which is converted into indoleacetic acid during extraction.
A simple technique of quantitative determination of IAA on TLC plates through colour reaction and densitometry has been brought out. For nanogram IAA amounts, specific radioimmunoassay (RJA) technique has been developed. High-resolution growth recorders for plant growth hormone studies have been developed and are of immense use.
Occurrence:
GLC and mass spectrometry have been used to confirm detection of IAA in some conifers like Pseudotsuga menziesii. The hormonal relationships in algae, levels of IAA in lateral buds, its presence in suspensor cells of Tropaeolum (0.7 ng/mg fresh wt.), its production by tomato embryos, role of IAA in fruit growth have also been worked out.
Dry grains of Avena have 8 mg/kg of IAA and only 5% of this is free whereas 95% is in esterified form. On the other hand, legume seeds have peptidyl IAA. In maize seeds bound auxins are esters between IAA and glucose, inositol and cellulosic glucan. In dry Avena seeds, IAA appears to be bound to heterogeneous polymer, probably gluco-protein.
IAA Transport:
Most available studies have shown polar transport of auxin by exchanges through the plasma membranes and the free space, plasmolyzed coleoptile segments where plasmodesmata were broken. IAA transport was intense as in the normal tissues. The general finding is that influx of IAA into the cell from the free space was passive, while efflux at the basal end of the plasma membrane depended on active processes.
Further pH gradients between the surrounding cell wall and its cytoplasm determine the auxin influx and efflux. Stray reports on acropetal IAA transport in isolated segments of coleptiles and stem was attributed to diffusion. On the contrary, basipetal transport involved active physiological processes.
The polar translocation is also sensitive to inhibitors viz., TIB A, ethylene. Further water stress also affects basipetal transport. The transport properties of auxin in roots and their possible role in geotropism is being discussed vigorously. All said and discussed it seems that the direction of IAA transport in roots depends mainly on the prevailing experimental conditions and the physiological conditions of the tissue.
Classically IAA transport is explained by a carrier-mediated, energy- dependent mechanism. However, more recent theory is based in differential pH of the cell interior and the solution bathing the cell wall having comparatively lower pH. This acid, enters the cell in the undissociated form and not as an anion.
Wilkins and his associates have demonstrated that polar transport of IAA took place predominantly in the stelar tissue of maize roots. However, in pea the transport occurred in the cambium and differentiated vascular tissue.
IAA has also been noticed in the phloem sap. It is generally assumed that pathway of IAA transport appears to be distinct from the polar transport system. It is still debatable how downward polar transport of IAA is accomplished.
Compounds like cyanides, 2, 4-dinitrophenol, which inhibit respiration, also inhibit polar transport suggesting that auxin transport is energy dependent process especially liked with oxidative metabolism of the mitochondria. Phytotropins chemicals like 2, 3, 5-triiodobenzoic acid (TIBA), morphactins, NP Appear to be specific inhibitors noncompetitive inhibitors of polar transport.
Thus, carrior-mediated transport through specific proteins is envisaged. Additionally diffusion may also be involved in auxin transport. The velocity of polar IAA transport has been estimated in the range of 5- 20 mm hr−1.
Metabolism:
The exact biosynthetic pathway of IAA is still debatable. Some investigations have even doubted the role of tryptophan as a precursor. Auxenic callus tissue in crown-gall tumour tissue from Vinca rosea, which is known to synthesize its own auxin, was shown to convert exogenously supplied indole and tryptamine to IAA but very little or no tryptophan is converted to IAA.
After its incorporation into plant tissues, most of the IAA is decarboxylated by lAA-oxidase, very little is degraded via the oxindole pathway or it may be converted to bound form. There are also suggestions that IAA amino acid conjugates were actually the physiologically active forms of IAA.
IAA Catabolism:
The available evidences suggest that IAA oxidase and peroxidase constitute the IAA-oxidising system and the activities of the two enzymes were situated in only one types of molecule. Some link between peroxidase, IAA-oxidase and polyphenol oxidase has also been suggested.
The association of three functions to a single molecule definitely is of immense physiological interest for the regulation of auxin level in plant tissues.
Nutrient conditions, toxic and deficient levels of manganese affect IAA oxidising system. Similarly a close correlation between the level of copper and IAA oxidase has also been demonstrated in some systems. Boron deficiency is also known to include IAA-oxidase. Likewise deficiency of zinc reported to stimulate oxidase activity in some systems. Auxin level can decrease due to photo-oxidation and its products are multiple.
The available data indicate two pathways of IAA catabolism, i, peroxidative decarboxylation, and oxidation to oxindole-3- acetic acid as below:
There is evidence that decarboxylation of IAA may take place. Detailed studies are needed to determine the relative significance of the two catabolic pathways.
IAA Conjugates:
In Zea mays kernels several endogenous IAA conjugates have been studied and these are esters of IAA and myo-inositol, wyo-inositol galactose, myo-inositol-arabinose, β1-4 glucan, etc. The formation of IAA conjugates is non-destructive and in some instances it is reversible. During seed germination they hydrolyzed to furnish free IAA.
The conjugates are easily transported and hydrolyzed at the appropriate place. Conjugates have several functions: storage form of IAA, act as transportable form during movement of IAA, and lastly IAA is protected against peroxidation. Moreover, formation of conjugates and hydrolysis possibly constitute a homeostatic mechanism for regulating the level of IAA in plants.
Growth Kinetics:
High resolution growth recording technique has been employed to show the overlapping of three separate responses. The first transient reduction of the growth rate had a lag phase of less than two minutes while the second transient indicated a sharp increase in growth rate induced by protons extrusion. The third increase was followed by a long lasting decline.
In the first phase a slight depolarization of about 5 mV lasted for 3 to 5 min. The other two reactions responded differently to cytokinins. CH and auxins, etc. These overlapping reactions occurred in many mono-and dicotyledonous species. In brief, it may be stated that before any biochemical studies of growth processes are demonstrated it is essential to elaborate growth kinetics.
Cell Wall and Plasma Membrane:
Most recent studies provide evidences on acid growth theory and also that proton extrusion was an important step in events leading to auxin-induced cell elongation. In elongating soybean hypocotyls it is reported that the external H+ ion concentration for pH 4.8 was adjusted to pH 5.4.
Additional of IAA stimulated elongation but did not adjust this pH. Apparently H+ ions stimulated elongation of hypocotyls but this reaction was different from auxin induced growth. In fact the auxin response had a longer duration.
The overall conclusion is that auxin affects cell wall loosening via H+ ions and proton extrusion was an important step in auxin induced growth. When Nojirimycin, a potent inhibitor of gluconase, was used it inhibited auxin induced growth but not H+ ion induced extension.
The simple conclusion is that auxin induces wall loosening in a way other than H+ ions. Epidermis definitely has a role for auxin induced elongation and proton extrusion.
Seemingly potassium functions as a counter ion when malate accumulation maintains the intracellular pH and auxin causes protons release. Some studies have also shown that auxin stimulates dark CO2 fixation into malate. In fact the first reaction of a cell to auxin is a change in the thickness of plasma membrane.
The response is rapid and reversible. Possibly microviscosity of the hydrocarbon region of the membrane increases and IAA causes a quick hyperpolarization of the electric potential between the vacuole and the external medium. Cleland is of the view that activation of electrogenic proton pump causes acidification of cell wall medium. In fact hyperpolarization stimulates influx of potassium ions.
The membrane permeability for potassium ions is affected by IAA and depends upon the Ca+ ions. Roots vary in their responses to auxin. H+ ions stimulate root growth. The recent thinking is that auxin increases ethylene production in roots but cannot explain auxin induced growth inhibition. Apparently the inhibitory effect of auxin is independent of auxin induced cell wall pH modification or ethylene production.
Binding Sites:
Large amount of data on auxin binding sites have accumulated recently. But a general conclusion still prevails that most information has come from the works of Ray (1977) who have provided evidence that in corn coleoptile a protein reversibly binds NAA was localized in the ER membrane.
A reducable group possibly a disulphide may be important for its role in binding. There is still a confusion whether binding site represents the receptor concerned with growth processes or transport or conjugation processes. In general, it is believed that the carrier involved in auxin transport is located in the plasma membrane and is the site of receptor for the transport inhibitor. The two situations are unreconcilable.
The auxin binding site is present in ER membrane while the primary site of auxin action is plasma membrane. Ray has suggested that the protein transport induced by the auxin receptor complex the ER may occur via the Golgi system. Some workers have shown two separate classes of binding sites for NAA and IAA.
These are called site I and site II. We do not have any information on the physiological responses of the two receptors. However, some attempts have been made to assign many amino acid residues at the binding site. Some workers have isolated a membrane rich fraction containing ATPase activity which specifically binds IAA.
Some investigators have advanced hypothesis on the structure-activity relationship of auxins and postulated that following attachment to a binding site the auxin molecule undergoes reorientation or conformational change.
Transcription and Translation Subcellular Structure:
IAA has been shown to stimulate RNA polymerase and thus the process of transcription. In fact, it is the polymerase I activity which is increased and this increased level seems to be correlated with the developmental stage of the tissue.
IAA affects this enzyme via.,“transcription factor” which is a specific protein. IAA also increases the quantity of polyribosomes which in turn enhance the rate of amino acid incorporation by them. Most recent studies have indicated that auxin affects at least three specific ribosomal proteins. It is quite likely that a shift in specific ribosomal proteins affects protein synthesis.
Mechanism of IAA Action:
When IAA is added to a plant system following set of events takes place:
Initially, IAA is adsorbed to a hormone-specific binding site to form an IAA-binding site complex. Then series of reactions are Initiated which comprise, membrane phenomenon causing media acidification; phenomena related to nucleic acids causing changes in enzymes concerned with morphogenesis. Both the events involve alterations in the wall plasticity resulting in growth.
Plasticity enhancement is dependent upon changes in the protein matrix of the cell wall, and changes in the cellulosic matrix and hemicellulosic matrix as shown below:
We shall discuss some of the steps briefly.
IAA Bindings:
Though still inconclusive, the general assumption is that the hormone reacts with a macromolecular cellular component to exert its influence. Several methods have been devised to isolate the binding site.
Membrane and Ion Movement causing Acidification of Media:
It has been shown beyond doubt that media acidification is somehow related to IAA-enhanced growth. The general assumption is that IAA activates a plasma membrane ATP, leading to the stimulation of active proton efflux from the cell.
Lowering of pH, in this way, causes activation of enzymes capable of hydrolyzing wall polysaccharides, softening of the wall and resulting in wall extension. It is also reported that isolated walls respond to low pH.
Media Acidification and its Effects:
The precise mechanism of media acidification is not clear, though the reaction has a requirement for Ca2+. Consequently, there is a decrease in the ATP/ADP ratio and enhanced respiration. ATP is also shown to restore IAA-induced growth under anaerobic conditions.
Undoubtedly media acidification is closely associated with IAA- induced growth, and appears to be a part of the growth phenomena. It is difficult to visualize how cell expansion, media acidification and ion movement integrate into a system which exhibits cell division and differentiation.
Change in Nucleic Acid and Protein:
Available evidences point out that IAA-induced growth is accompanied by enhanced synthesis of both nucleic acid and protein. Perhaps in the coming years it would be possible to determine the enzymes which are activated following treatment with IAA. In any case, IAA triggers series of events which cause cell extension and alterations in RNA and protein.
Changes in Wall Plasticity:
As the protoplast enlarges, cell wall stretches or grows. In fact recent view is that mechanical properties of wall are an integral process concerned with growth though wall itself is not the primary target of IAA.
The plant cell wall is a highly ordered organelle having complex cellulosic microfibrils held in a matrix of hemicellulose and protein. With the addition of IAA, the solution in which the cell is bathing becomes acidic causing cell wall deformity.
This is due to enzymatic changes in the wall. It is not clear whether such an increase of enzymes is due to enhanced activity of enzymes or due to added secretion of enzymes into the wall.
Functions:
Fig. 20-4 and Table 20-1 give the summary of physiological effects of auxin.
Some points are discussed below:
IAA and Ethylene Formation:
IAA has been shown to stimulate ethylene formation in several tissues. IAA and also ethylene cause epinasty. IAA and ethylene effects are opposite in abscission, senescence, etc. Like IAA ethylene also inhibits root growth and some workers attribute auxin effect on root geotropism due to ethylene formation in roots. It has been suggested that auxin action in roots is mediated through ethylene.
IAA Effects on Specific Enzymes:
IAA enhances citrate-condensing enzymes in one system and inhibits pyruvate carboxylation enzymes in another. IAA inhibits indole ethanol oxidase. It affects the activity of cellulase, lipase, invertase, peroxidase, IAA-oxidase, etc.
Effects on RNA and Protein Synthesis:
In several systems IAA enhanced RNAs and the increased protein synthesis accompanying growth in a system could be attributed to the increased RNA synthesis. Hormone function decreased following the use of nucleic acid and protein synthesis inhibitors. Influence of IAA is on all three types of RNAs.
It may be stabilizing mRNA; it may be a signal for polypetide, chain initiation or IAA may act like essential amino acids needed for tRNA synthesis. It may be acting at the level of a regulator or may even be activating transcription. Perhaps there are several primary sites of action of auxin. It may be affecting cell wall or plasma membrane.
Stimulation of Cell Division:
In cambium and also cell division during root formation in cell tissue culture studies also have shown that IAA promotes both cell division and cell elongation.
Apical Dominance:
It is generally observed that the development of the lateral shoots is inhibited by a substance which arises from the “apex”. Once the tip of the main shoot is removed, the side shoots develop. This is referred to as apical dominance.
The removal of main shoot when replaced by IAA, the development a lateral shoots is inhibited again. Obviously, IAA is one of the factors which causes this dominance. In recent years, it has been shown that the inhibition of lateral shoots development is caused by ethylene and IAA induces the formation of the latter.
The role of IAA in the inhibition of leaf shedding, inducing fruit formation in several plants like apples, cucumbers, tomatoes, promoting respiration or propagation of plants by stem cuttings, thinning of flowers and fruits ; controlling preharvest fruit drop, etc. is well known.
IAA has also been shown to affect the activitiy of several enzymes (cellulase, lipase, invertase, peroxidase).
Antiauxins:
There are some substances which have been shown to limit the action of auxin. These are called anti-auxins. The concept of anti-auxin includes chemicals which resemble auxin closely but lack at least one requirement for activity.
Normally auxin has three structural requirements whereas anti-auxin activity might arise from an inadequate acid side chain or unsaturated ring or insufficient space relation between the two. 2, 3, 5-TIBA is a well-tested anti-auxin and analogues of 2, 4-D, 2, 4, 5-T also have been shown to have anti-auxin activity.
It may be stated that spatole and N-acetyl indole acetic acid are not effective anti-auxins. However, much remains to understood as regards the concept of ‘anti-auxins’.
Several of the synthetic auxins have also been reported and some of these include 2 4-D, a- napthylacetic acid. 2, 4, 5-T, etc. On the other hand T1BA is “antiauxin”. 2, 4-D (2, 4- dichlorophenoxyacetic acid) in high concentrations is a potent weed killer especially dicotyledons. However, monocotyledons including cereals when sprayed with this are not inhibited. Thus, this compound is used in eradicating dicotyledonous weeds.
Some of the synthetic auxins are more effective than IAA which occurs naturally. Possibility is that synthetic auxins resist oxidation by IAA oxidase in the natural system.
Phenylacetic acid and chlorinated IAAs are two of the naturally occurring auxins other than IAA. Of the two, the latter appears to be made by chloroperoxidase or peroxidase and is more active than IAA. However, phenylacetic acid is usually less active than IAA and is derived from phenylalanine.