In this article we will discuss about Auxins. After reading this article you will learn about: 1. Structure of Auxins 2. Biosynthesis of Indole-3-Acetic Acid 3. Responses of Plants 4. Mode of Action 5. Bioassay Methods.
Contents:
- Structure of Auxins
- Biosynthesis of Indole-3-Acetic Acid
- Responses of Plants to Auxins
- Mode of Action in Auxins
- Bioassay Methods in Auxins
Contents
1. Structure of Auxins:
Indole acetic acid (IAA) is the principal native auxin of higher plants. There is a ring structure and a side chain.
In indole auxins, the ring is indole (i.e., benzopyrrole) and the side chain is a fatty acid attached to carbon 3 of pyrrole part of indole. When the side chain is acetic acid, the auxin is indoIe-3-acetic acid. Similarly, the side chains of indole-3-butyric acid (IBA) and indole-3-propionic acid (IPA) are represented by butyric acid and propionic acid, respectively.
In addition to IAA, IBA and IPA, a large number of indole compounds with auxin activity have been identified from plant tissues.
These are as follows:
IndoIe-3-acetaldehyde (IAAld), indole-3-pyruvic acid (IPyA), indoIe-3-ethanol (IEtOH), indole-3-glycolic acid, indole-3-acetonitrile, indole-3-carboxylic acid, ethyl indole-3-acetate, indole-3-acetamide, indole acetylaspartic acid, indole acetyl glutamic acid, IAA glucosyl ester, tryptamine, indole-2,3-dione (isatin).
Most of these compounds either occur in the pathway of auxin biogenesis or are among the breakdown products of IAA and its precursors having weak or no auxin activity.
(a) Bound Auxins and Auxin Precursors:
Many experiments have given rise to the concept of bound auxin which is neither diffused nor transported.
These are thought to be storage products or auxin precursors from which true auxins may be liberated. There are a large number of such binding forms, such as auxin bound to protein, to RNA and to inositol. Indole acetyl inositol may be regarded as an auxin precursor from which active auxin may be liberated with dilute alkali.
In addition, a molecule of arabinose may be attached to inositol. A thioglucoside of indole-3-acetonitrile (IAN) named glucobrassicin has been discovered in crucifers which upon enzymatic hydrolysis by myrosinase yields thiocyanate and IAN. Its methoxy derivative called neoglucobrassicin has also been obtained.
In presence of ascorbic acid, the hydrolysis leads to the formation of IAA-ascorbic acid complex known as ascorbigen. Other examples of bound auxin are indole aspartic acid, indole glutamic acid, indole glucoside and indole rhamnoside.
Besides indole rings, other types of ring may also be present. Phenoxy rings have given rise to a class of compounds having herbicidal properties, for example, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).
Examples of synthetic auxin with naphthalene ring are naphthalene acetic acid (NAA) and naphthoxy acetic acid (NOXA). A simple benzene or phenyl ring may serve as the ring of auxin molecule, for example, phenylacetic acid and derivatives of benzoic acid.
(b) Structure-Activity Relationship:
Three structural requirements must be fulfilled by a molecule having auxin activity.
These are:
(i) unsaturated ring
(ii) acidic side chain of an optimum length
(iii) spatial relation between the two. A 5.5 A spacing is necessary between the charge of carboxyl (-) and the ring charge (+) on the molecule of auxin.
(c) Synthetic Auxins:
Many synthetic compounds may be called auxins because they exhibit physiological action similar to that of IAA. Since these compounds are not naturally occurring they may be called growth regulators instead of hormones. There are five major groups of synthetic auxins.
These are:
(i) Indole acids like indole pyruvic acid (IPA) and indole butyric acid (IBA). However, these two compounds are not altogether synthetic because each has been shown to occur in a few plants,
(ii) Compounds with phenoxy ring like 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) which have herbicidal properties,
(iii) Compounds with naphthalene and naphthoxy rings are naphthalene acetic acid (NAA) and naphthoxyacetic acid (NOXA),
(iv) Compounds with simple benzyl or phenyl ring and derivatives of benzoic acid as the ring structures have given rise to synthetic auxins like phenylacetic acid, 2,3,6- and 2,4,6-trichlorobenzoic acids and 2-methoxy-3,6- dichlorobenzoic acid (dicamba), the latter being used as strong weedicide,
(v) These are picolinic acid derivatives. One of the most powerful herbicides known as Picloram or Tordon (4-amino-3,5,6- trichloropicolinic acid) belongs to this group.
An example of synthetic auxin with chlorophenoxy ring is 2-methyl-4-chlorophenoxyacetic acid (MCPA).
(d) Auxin Transport:
The influences of auxin on plant growth and development are correlated with polar movement of endogenous auxin. In shoot tissues, the polarity of auxin transport is basipetal, i.e., auxin moves from morphologically apical to basal region regardless of whether the base is normally down or turned with the apex down.
In root also the transport is polar but it is acropetal, i.e., from the base of the stem to the root tip. Polar transport is an energy-requiring process. Anaerobic condition and metabolic poisons such as cyanide and dinitrophenol inhibit polar transport. Polar transport occurs through parenchymatous cells, associated with or differentiating into vascular tissue but not through the phloem and xylem.
Quite a number of hypotheses have been proposed for understanding the mechanism of polar transport of auxin. Scrank (1951) showed differences in electric potential from apex to base of coleoptiles. Again, the shaded side became electropositive under unilateral illumination as opposed to the lighted side.
When geotropically stimulated, the lower side of horizontally laid coleoptile became electropositive. It was shown that auxin which exists as an anion (A–) would move along the electric gradient towards the positive side. Newman (1963), on the other hand, could not confirm the electric potential gradient as the cause of polar auxin movement.
He postulated that an electric wave is produced during auxin movement down a coleoptile and an electric field thus set up may in turn push the hormone further down the coleoptile. Leopold and Hall suggested that a secretion mechanism causes the movement of auxin from the basal end of cells and a basally-located active secretion pump has been proposed to account for the polarity.
Goldsmith (1977) and Rubery (1987) have now simplified this model by the chemiosmotic hypothesis of polar auxin transport in which secretion has been described as energetically downward auxin anion efflux. This model assumes that a carrier protein which is responsible for the efflux of auxin anions is located preferentially at the basal ends of cells in the transport pathway.
This would result in bringing a low auxin accumulation rate at the basal end with more than anion efflux sites than at the apical end. Thus, an uphill concentration gradient of auxin can be established to account for basipetal polarity. Thus, according to the chemiosmotic polar diffusion hypothesis, the energy stored in an electrochemical gradient set up by cellular accumulation of anxin can be used for polar transport.
2. Biosynthesis of Indole-3-Acetic Acid:
Indole-3-acetic acid (IAA) is structurally related to the aromatic amino acid tryptophan. It is considered that tryptophan is the probable precursor of IAA.
Plants convert tryptophan to IAA by several pathways which are described below:
(a) Indole Pyruvic Acid (IpyA) Pathway:
The first step involves transamination or deamination of TPP by TPP aminotransferase. Second enzyme is IpyA decarboxylase by which IpyA is converted to lAAld. The last step is catalyzed by IAAld dehydrogenase or oxidase in which lAAld is oxidized to IAA. This pathway is known as indole pyruvic acid pathway.
(b) Tryptamine (Tam) Pathway:
This pathway is known as tryptamine (Tam) pathway. First reaction is catalysed by TPP decarboxylase by which TPP is converted to tryptamine. Then tryptamine is deaminated by tryptamine deaminase to indole acetaldehyde (lAAId), which is then oxidized to IAA either by an oxidase or a dehydrogenase. Thus, the indole acetaldehyde (lAAId) is the immediate precursor of indole acetic acid (IAA) by both routes.
(c) Indole Acetaldoxime (lAOx) Pathway:
In this pathway, tryptophan (TPP) is first converted to tryptamine (Tam) by decarboxylase reaction. Then by oxidation of this primary amine, Tam will be converted to lAOx by means of a mono-oxygenase. In this case, one atom of the oxygen molecule is directly inserted into the product IAOx and the other atom of oxygen is reduced to water.
IAOx is converted to indole acetonitrile (IAN) by IAOx hydro-lyase in which H2O is removed. Then nitrilase enzyme acts on IAN in a two-step reaction. In the first step, one molecule of water is added to IAN and indole acetamide (1AM) is formed as an intermediate. In the second step, the second water is added to IAM and IAA is produced together with NH3.
The above pathway is found in non-Cruciferous plants. In Cruciferous plants, IAOx is converted to glucobrassicin, a thioglucoside of IAOx, by an additional reaction with thioglucose (SGIu) and sulphate, in which the intermediate compound is desthioglucobrassicin (GluBr). Then GluBr is acted upon by myrosinase and IAN is produced which is ultimately converted to IAA by nitrilase.
(d) Tryptophol (TOL) Pathway:
It is actually a modification of the first, i.e., IPyA pathway. The first and second reactions are similar to those of IPyA pathway, i.e., deamination followed by decarboxylation. The last reaction, which involves oxidation or reduction of lAAld, may engage either dehydrogenase or oxidase or dismutase.
It is possible that metabolism of IAAld to IAA and TOL may be catalysed by a dismutase, i.e., coupled enzyme reactions involving pyridine nucleotides leading to oxidation to form IAA and reduction to form TOL. On the other hand, two separate dehydrogenases, viz., acetaldehyde dehydrogenase and alcohol dehydrogenase, may also function. In addition, TOL oxidase has also been identified (TOL → IAAld).
In some plants, IPyA may undergo reduction to indole lactic acid (ILA) by IPyA reductase. It has been suggested that in some cases, ILA acts as the direct precursor of TOL through ILA decarboxylase. Since ILA decarboxylase has not generally been detected and isolated, this conversion (ILA → TOL) seems unlikely.
(e) Tryptophan-Independent Pathway of Auxin Biosynthesis:
In some plants, IAA is also synthesized either from indole or from indole-3-glyceroI phosphate. In studies with Arabidopsis mutants, which are blocked in tryptophan biosynthesis, it has been shown that IAN accumulates to much higher level compared to the wild type.
In tomato, IPyA has been shown to be synthesized without the involvement of tryptophan. Depending on the species, either IAN or IPyA may be synthesized from either indole-3-glycerol phosphate or indoles respectively, which are ultimately converted to IAA. The suggested branch points from tryptophan-dependent pathway are at IGP or at indole, whereby IAN and IPyA are two possible intermediates.
3. Responses of Plants to Auxins:
(a) Cell Enlargement:
Auxins induce rapid cell elongation in isolated stem and coleoptile sections. The time-course of auxin-induced cell enlargement shows that there is a lag of at least eight minutes before the growth. Then the rate rises until a maximum of 5-10 fold is reached after 30-60 min which remains constant for hours or even days.
Initiation of auxin-induced growth requires the continued presence of auxin, a continued supply of ATP and active ATPase’s, protein synthesis and sufficient cell turgor so that the cells are in a state of tension.
Although the initial rate of auxin-induced growth is independent of the presence or absence of absorbable solutes like sucrose, but continual rapid elongation requires sucrose.
Since auxin-induced cell enlargement is an energy-requiring process, all inhibitors of ATP synthesis (e.g., KCN, DNP, and azide) or of ATPase activity (e.g., vanadate, DCCD, DES) block the process. All inhibitors of protein and RNA synthesis likewise inhibit auxin-induced growth.
(b) Cell Division:
In an intact system, auxin stimulates cell division in the cambium and formation of new phloem and xylem. In cultured tissues, auxins usually in combination with cytokinins promote cell division. In tobacco pith system, suitable auxin-cytokinin ratios can affect organ formation. For instance, in tissue culture, when auxin level is higher than cytokinin, roots form.
When cytokinin is higher than auxin, shoots are produced. When the concentrations are about the same, a callus mass is produced. Thus by adjusting auxin-cytokinin ratios, either shoot or root formation can be initiated.
(c) Vascular Tissue Differentiation:
The vascular system is composed of two kinds of conducting tissue, viz., the phloem through which organic materials are transported and the xylem which is the path of transport of water and soil nutrients. Auxin indole-3-acetic acid (IAA) is the main limiting and controlling factor for both phloem and xylem differentiation.
One of the major signals produced by the young leaves is auxin which moves in a polar fashion towards the roots. Vascular tissue differentiation occurs along the polar flow of auxin from leaves to roots. There is evidence that low auxin levels induce phloem with no xylem whereas both xylem and phloem differentiations take place at higher auxin levels.
(d) Root Initiation:
Root initiation is generally regulated by auxin. The localization of root generation at the basal end of stem cuttings is due to polar movement of auxin toward the physiologically lower end that was nearer the root tip of the intact plant.
Synthetic auxins like NAA and IBA added from outside to the root end of the freshly excised stem cutting have been found to initiate root initiation and thus can be used as rooting hormones in horticultural practice.
(e) Tropic Responses:
Auxin mediates the tropistic (bending) response of shoots and roots to light (phototropism) and to gravity (gravitropism). Shoots grow toward unilateral light sources showing positive phototropic response. Roots generally bend away from unilateral light sources and are negatively phototropic.
In the phototropic response mechanism of higher plants, the Cholodny-Went hypothesis says that auxin becomes asymmetrically distributed with more accumulating on the shaded side than on the illuminated side and this produces the bending response.
The prostrated seedlings lying in a horizontal manner are said to be gravitropically (geotropically) stimulated whereby shoots and roots show opposite gravitropic curvature.
The Cholodny-Went hypothesis says that hormone asymmetry which is the essential component of the process results from basipetal transport of IAA from the upper to the lower sides of gravistimulated roots and shoots whereby cell elongation is inhibited in the former and stimulated in the latter.
Nowadays, the Cholodny-Went hypothesis, however, is open to question and has been challenged as the sole mechanism for explaining both types of tropic responses.
(f) Apical Dominance:
The concept of apical dominance is based on domination of the apical region of shoot axis over the lateral buds which are also termed correlative inhibition. In a broad sense, this means complete or partial inhibition of initiation and development of lateral (usually axillary) buds by an actively growing apical region.
IAA formed in apex has distinct role in lateral bud inhibition. Surgical experiments have shown that the shoot apical region appears to be the main source of lateral bud inhibition.
It has been shown that removal of the tip portion of shoot (i.e., decapitation) releases apical dominance and consequently lateral bud formation is stimulated. Auxin applied on decapitated stump acts to replace the tip and appears to re-establish apical dominance and prevents lateral bud growth.
The phenomenon of apical dominance is broad ranging, encompassing at least four themes:
1. Complete or partial inhibition of development of lateral (usually axillary, but occasionally adventitious) buds by an actively-growing apical region on the same or a different shoot axis.
The apex may also control the initiation of buds. Domination of the apical region over the lateral buds is termed ‘correlative inhibition’. It is seen especially in etiolated shoots but also in leafy and modified shoots (tubers, rhizomes, stolons, bulbs, corms, etc.).
2. The suppressive influence of one dominant shoot upon one or more subordinate shoots.
3. Influence of the apex on the development and positioning of leaves, axillary shoots, stolons, tubers, rhizomes and roots. An inflorescence apex can modify the positioning and development of the floral and derived structures.
4. Influence of the apex on the transport of nutrients and cellular differentiation in the stem or root axes. Thimann and Skoog originally proposed that auxin from the shoot apex inhibited the growth of the axillary bud directly. This is called direct-inhibition theory. According to the theory, the optimal auxin concentration for bud growth is much lower than the auxin concentration normally found in the stem.
The level of auxin normally present in the stem was thought to inhibit the growth of lateral buds. If the direct-inhibition model of apical dominance is correct, the concentration of auxin in the axillary bud should decrease following decapitation of the shoot apex.
However, the reverse appears to be true. This was demonstrated with transgenic plants that contained the reporter genes for bacterial luciferase (LUXA and LUXB) under the control of an auxin-responsive promoter. These reporter genes allowed for the study of the level of auxin in different tissues by monitoring the amount of light emitted by the luciferase-catalysed reaction.
When these transgenic plants were decapitated, expression of the LUX genes increased in and around axillary buds within 12 hours. This experiment indicated that after decapitation, the auxin content of the axillary buds increased rather than decreased. Direct physical measurements of auxin levels in buds have also shown an increase in the auxin of the axillary buds after decapitation.
The IAA concentration in the axillary bud of Phaseolus vulgaris increased five-fold within 4 hours after decapitation. These and other similar results make it unlikely that auxin from the shoot apex inhibits the axillary bud directly.
Snow developed the correlative inhibition or indirect theory, the gist of which is that as a result of auxin induced growth in the apical bud an inhibitor is formed which moves into lateral buds and suppresses growth. There is little evidence to support the theory.
Went suggested nutrient diversion theory in which endogenous and exogenous auxin create an attraction of nutrients towards the point of auxin synthesis (the active apex) or application, such that the lateral buds become deprived of nutrient supply and do not grow.
The vascular connection theory is a combined hypothesis blending the elements of the ‘direct inhibition’ and ‘nutrient diversion’ theories.
It explains that the auxin and the correlative inhibitors prevent the entry of factors into the lateral buds by an effect on the vascular connections between bud and the stem. The effect may be on the vascular differentiation. Recent analyses indicate that a correlation does not always exist between vascular differentiation and the loss of the quiescent state.
Phillips developed the hormone balance theory explaining that a balanced hormone composition controls the inhibition and stimulation of development. Evidence in favour of this theory is fragmentary.
All of the plant growth substances directly or indirectly affect bud growth, and there is evidence to suggest that IAA, cytokinin and in some cases ethylene could have basic roles in correlative inhibition. Till date none of the above theories is satisfactory.
(g) Leaf and Fruit Abscission:
In the intact plant, IAA present in leaf blade inhibits its abscission but when the supply of IAA becomes low either by normal aging or by artificial de-blading abscission occurs. In the isolated system, a de-bladed petiole is found to abscise faster. In de-bladed leaves, IAA when applied to the proximal end of the petiole can substitute for leaf blade and retard abscission of the petiole.
It is a common experience that mature fruits of apple, pear, lemon and grape are frequently found to drop before the time of commercial harvest resulting in lower quality fruits. The auxins like 2,4,5-trichlorophenoxy propionic acid (2, 4, 5-TP) and the dichlorophenoxy analogs (2, 4-DP), NAA, 2, 4-D, when applied during the mid-states of fruit growth have been found to prevent abscission of mature fruit.
The role of auxin in fruit abscission is rather complex. The auxins are frequently used both for the prevention of fruit drop as well as for the chemical thinning of young fruits. It is a common orchard management practice that the fruit-growers tend to remove excessive numbers of young fruits.
(h) Fruit Setting, Fruit Growth and Fruit Ripening:
An auxin like 4-chlorophenoxyacetic acid (4-CPA) is used to increase fruit set and growth of tomato.
Although no clear relation has been found between fruit ripening and endogenous auxin content, it has been suggested that ripening may be related to a decline in auxin. An auxin like 2,4-D has been shown to cause a dual effect on ripening, viz., stimulates ripening through ethylene production but may also cause delay in ripening.
(i) Flowering:
Auxins like NAA and IAA promote flowering in the members of the Bromeliaceae and this effect is due to auxin-induced ethylene production. Nevertheless, the proposed role of auxin in the control of flower formation is not universal since auxin application tends to be inhibitory to flower formation in most plants. It now appears that these inhibitory effects are also due to auxin-induced ethylene production.
(j) Assimilate Movement:
Hormones in general are involved in the regulation of source-sink relations. The maximum amount of hormones occurs in seeds during the time of rapid dry-matter accumulation. It appears that assimilate movement is enhanced towards an auxin source possibly by an effect on phloem transport.
(k) Changing Sex Expression:
Perfect flowers are those which contain both stamens and pistils. In Cucurbits like Cucumis, perfect flowers are initiated but one sex organ fails to develop which leads to the development of monecious plants having staminate and pistillate flowers. Application of auxin to flower buds at the bisexual stage leads to the formation of female flowers. It now appears that IAA acts through ethylene in this process.
4. Mode of Action in Auxins:
Auxin has been shown to influence a variety of growth and developmental responses including cell elongation, cell division and cell differentiation. In the initial work performed in 1950s, it was shown that IAA markedly increase nucleic acid contents (both DNA and RNA) in plant cells within 48 hours.
This observation led Silberger and Skoog to suggest that auxin influences nucleic acid synthesis. This hypothesis known as gene activation or gene expression hypothesis was no doubt very attractive since it could suggest a general mechanism of auxin action for diverse responses.
Later it was discovered that auxin effects on nucleic acids were only indirect. Actually, IAA was shown to act at various stages on different factors concerned with DNA replication, transcription and translation—thus all the aspects of molecular biological phenomena have been shown to be influenced by IAA.
(a) Auxin Interaction with DNA and Protein:
There are evidence that auxin may interact with DNA or chromatin directly or after complexing with some receptor proteins. Mathysse and Philips (1969) first showed that the synthetic auxin 2,4-D stimulates RNA synthesis by chromatin via a protein fraction.
This observation has been supported by Mondal et al. who showed the existence of an IAA-receptor complex which enhances DNA- dependent RNA synthesis in the presence of RNA polymerase, initiation factor B and DNA.
Auxin-binding proteins have been isolated by Biswas and his co-workers (1982) from Avena roots and coleoptiles, nuclei of tobacco cells, plasma membrane of soybean cotyledons and cytosols of pea, corn and tobacco pith cells. The molecular weights of these proteins vary between 20,000 and 2,00,000.
(b) Nucleic Acid and Protein Synthesis:
In soybean hypocotyls treated with high concentrations of auxin, there are dramatic increase in RNA, DNA and protein contents. Increase in RNA content mainly results from an increase in ribosomal RNA, which leads to an increase in the pool of ribosomes. Both the rate of RNA chain initiation and chain propagation by RNA polymerase are increased within 24-28 hours of auxin application.
It has been shown that auxin induces de novo synthesis of RNA polymerase I, the enzyme involved in transcription of rRNA genes. Furthermore, the increase in rRNA and ribosome accumulation have been shown to be correlated with a large-scale increase in translatable messenger RNA (mRNA).
The activity of RNA polymerase II which is involved in the transcription of mRNA genes also increases following auxin application and the amount of the enzyme also increases a little by de novo synthesis. When sections of elongating zone of hypocotyl are incubated in the presence of auxin, rapid cell elongation is observed.
Since RNA and protein synthesis are considered as integral part of growth by cell elongation, studies were made on the effect of RNA synthesis inhibitor, actinomycin D and protein synthesis inhibitor, cycloheximide on the process.
It was demonstrated that in the presence of such inhibitors, auxin-induced elongation ceases which implies that the ability of auxin to increase cell elongation is dependent upon continued RNA and protein synthesis.
In order to determine the primary action of auxin, the gene activation hypothesis was invoked to explain the process of cell elongation.
Since there is a very brief lag between auxin application and cell elongation, it was proposed by Masuda and his associates that auxin induces specific mRNA during this lag and this mRNA produces the enzyme, β-1,3- glucanase participating in cell wall hydrolysis leading to wall loosening and increase in plasticity.
They have presented a model—auxin primarily acts on primer DNA producing specific mRNA which codes for the enzyme, glucanase.
Then it was argued that if the initial response to added auxin was the de novo synthesis of RNA and proteins necessary for cell enlargement, these macromolecules would have to be synthesized during a period of about 10 minutes before an increase in elongation is observed.
From kinetic studies on elongation rate, it was shown that RNA synthesis does not occur in many organs for 10-60 minutes after auxin addition and it was concluded that the initial auxin action is probably not at the level of gene activation. It was suggested that the initial action of auxin was to cause cell walls to loosen.
When it was proposed that the factor responsible for this wall loosening was hydrogen ions, the acid-growth or media acidification or cell wall acidification hypothesis was formulated.
(c) Identification of a Possible Auxin Receptor:
According to the acid-growth hypothesis proposed to explain the mechanism of auxin action, the role of protein extrusion caused by plasma lemma-bound ATPase proton pump was emphasized.
This suggests that the auxin receptor is located on the plasma membrane. Contrary to this original assumption, auxin-binding sites were subsequently identified in the endoplasmic reticulum (site I), the plasma membrane (site II) and the tonoplast (site III). But recent work has pointed out that the binding site is thought to be only associated with site I, i.e., endoplasmic reticulum.
The protein responsible for site I auxin binding in the ER has recently been purified and named ABP 1 (auxin-binding protein 1). This protein consists of a 22kD polypeptide that forms a dimer in its native state. A single ABP 1 gene is present in Arabidopsis, whereas maize has five genes.
(d) Auxin and H+ Ion Pump:
If auxin causes the cell wall and the medium to become more acidic, it can achieve this by causing protons, i.e., H+ ions to leave the protoplasm and enter the wall by outward pumping of protons. According to Hager et al., cell wall growth is due to plasma membrane-bound ion pump which cause extrusion of H+ ion from cytoplasm into the wall.
As most ion pumps are ATPase’s, ATP must be required for proton efflux and the effect is enhanced by Mg2+ and K+ ions (Fig. 13.4). According to this hypothesis, IAA acts as an effector of membrane-bound ATPase proton pump which will be operative and will be able to transport H+ ions when combined with auxin.
(e) Acid-Growth Hypothesis:
Cleland (1971) suggested that the initial action of auxin was to cause cell walls to loosen. When it was proposed that the factor responsible for this wall- loosening was hydrogen ions, the acid-growth hypothesis, also known as media acidification or cell wall acidification hypothesis was formulated.
According to the acid-growth hypothesis of auxin action, promotion of cell elongation by auxin is due to the stimulation of H+ ions efflux from the cytoplasm into the cell wall. Acidification of the wall is thought to enhance wall loosening and allow rapid growth.
It was further observed that if isolated stem sections are placed in acid solution (about pH 3.5), they elongate even in the absence of auxin. Rayle and Cleland named this as acid growth effect as opposed to ‘auxin growth effect’.
From the similarities of effects of auxin and acid pH, two possibilities are likely (i) auxin directly breaks acid-labile bonds (mostly hydrogen bonds) in walls through a non-enzymatic process, or (ii) auxin causes wall to become more acidic, thereby breaking the acid-labile bonds indirectly via an enzyme requiring low pH, so this is an enzymatic effect.
Thus, it was suggested that appearance of nucleic acids followed by binding with the are later events of auxin action when cells have already started elongation.
It was concluded that the site of action of auxin is on some pre-existing cellular systems containing cell-wall hydrolysing enzymes leading to their allosteric activation for the hydrolysis of cell- wall polymers. This means that control by auxin at the transcriptional and translational levels is not ruled out but these are considered as secondary effects.
Then the question arises, “Does auxin directly interact with the plasma membrane (PM)- associated ATPase?” .In this respect, in vitro effects of auxin on isolated ATPase’s have been inconsistent and confusing. In general, stimulation of isolated ATPase’s by auxin have been quite small in magnitude occurring over restricted auxin concentrations which is difficult to reproduce.
In corn coleoptiles, auxin has not been found to bind to PM-ATPase’s. Thus, the site of ATP action is thought to be somewhere else than the plasma membrane. So, it will appear logical to infer that auxin does not directly activate a PM-ATPase.
An important clue to the mechanism of auxin-induced proton efflux may come from the effect of protein synthesis inhibitors on the process, which cause rapid (< 5min) and almost total inhibition of H+ efflux. This suggests that a continued protein synthesis is necessary for the operation of the proton pump.
This observation can be explained in two possible ways:
(1) Auxin-sensitive ATPase’s may be markedly unstable and hence labile, so these would require continued synthesis.
(2) The second possibility is that the requirement is not for any specific protein, but for protein synthesis in general as a process. For example, protein synthesis is thought to consume some compound (e.g., GTP), which acts as a negative effector for the ATPase.
Thus, in the presence of a protein synthesis inhibitor, say cycloheximide (CH), GTP will tend to accumulate owing to its non- consumption in protein synthesis. Hence GTP will act as an inhibitor of ATPase and proton efflux is eventually inhibited.
A new hypothesis has been put forward by Cleland regarding the possible involvement of a second factor in the modulation of PM-ATPase. It may be assumed that inhibition of protein synthesis would permit the factor (e.g., GTP) to build up thus inhibiting ATPase.
Then the question is, “What role might auxin play in such a scheme?” The answer is that auxin does not have to act at the plasma membrane, but once it is taken up into the cell, it might interact with some soluble enzyme system in such a way as to alter the level of the negative effector (factor f) or direct it towards protein synthesis (Fig. 13.4).
The acid growth hypothesis allows the following predictions:
1. Application of acid buffers should promote short-term growth.
2. Auxin should increase the rate of wall acidification through proton efflux, and the proton extrusion kinetics should correlate with auxin-induced growth.
3. Auxin-induced growth should be inhibited by neutral buffers.
4. Compounds other than auxin promoting proton extrusion should stimulate growth.
All of these predictions have been confirmed. Auxin stimulates proton efflux into the cell wall after 10 to 15 minutes of lag period, which is consistent with the growth kinetics. Auxin-induced proton extrusion probably involves both activation as well as de novo synthesis of H+ -ATPase’s.
Auxin could increase the rate of proton extrusion by two possible mechanisms:
1. Activation of pre-existing PM H+ -ATPase’s.
2. Synthesis of new H+ -ATPase’s, which are secreted and ultimately integrated into the plasma membrane.
(i) H+ -ATPase activation:
When auxin was administered to isolated membrane vesicles from tobacco cells, about 20% stimulation of the proton-pumping activity was observed, indicating smaller activation of the pre-existing H+-ATPase’s.
More stimulation (about 40%) was evident if the living cells were treated with IAA prior to the isolation of the membrane, suggesting the requirement of a cellular factor too. Various auxin-binding proteins (ABPs) have been isolated and appear to be able to activate the plasma membrane H+ -ATPase’s in the presence of auxin.
(ii) H+ -ATPase synthesis:
The inhibition of protein synthesis by cycloheximide even in the presence of IAA suggests that auxin might also stimulate proton pumping by increasing the synthesis of the H+ -ATPase.
An increase in the amount of PM-ATPase in maize coleoptiles was detected after only 5 minutes of auxin treatment, and after 40 minutes the amount reached to double. It has been reported that a threefold stimulation of H+ -ATPase mRNA synthesis by auxin took place specifically in the nonvascular tissues of the coleoptiles.
(f) Auxin Alters Gene Expression:
It was initially believed that rapid auxin action like cell elongation did not involve an altered pattern of gene expression.
The arguments made in late 1960s and early 1970s relied mainly on the kinetic studies on elongation induced by auxin, which showed that RNA synthesis did not occur before 5-10 minutes when cell elongation had already taken place. Based on this observation it was concluded that altered gene expression was not implicated in rapid auxin action.
With the advances in the techniques in molecular biology of late 1970s and early 1980s, it is now recognized that auxin can stimulate the expression of certain genes with a lag time of less than 5 mins.
It has become possible to detect rapid changes in the levels of translatable mRNA, which are able to initiate protein synthesis. Studies made by McClure et al. have shown that auxin can stimulate the transcription of specific mRNAs within 5 minutes.
This suggests that gene expression may indeed be involved in the rapid growth response to auxin.
When auxin binds to its receptor, a selected group of transcription factors are activated, and these activated factors enter the nucleus, and promote the expression of specific genes. Primary response genes or early genes may be defined as those genes whose expression is stimulated by the activation of pre-existing transcription factors.
Eventually, the early genes may be expressed within a very short time lag. On the contrary, the transcription of secondary response genes or late genes is required for the long-term slower responses to auxin. Thus, it seems likely that gene expression plays a role not only in slow effects but also in rapid effects of auxin.
Auxin regulates the expression of early genes through the following transcriptional activation steps:
1. Normally, the auxin response factor (ARF), forms inactive heterodimers with AUX/ IAA proteins coded by AUX/IAA gene preventing transcription of the early auxin genes.
2. In the presence of auxin, AUX/IAA proteins are targeted for destruction by an activation of ubiquitin ligase (ubiquitinization pathway).
3. The AUX/IAA proteins tagged with ubiquitin are then degraded by the proteasome (a proteolytic enzyme).
4. This IAA induced degradation of the AUX/IAA proteins allows the formation of active ARF homodimers.
5. The active ARF homodimers then bind to palindromic auxin response elements (AuxREs) in the promoter regions of the early genes, activating transcription.
6. Transcription of the early genes initiates the auxin-induced growth.
There are five major classes of early genes stimulated by auxin.
These are Aux/IAA gene family, the SAUR (small auxin up-regulated RNAs), producing a group of auxin-stimulated mRNAs, GH3 gene family, genes that encode glutathione S-transferase-like (GST-like) proteins, and genes that encode 1- aminocyclopropane-1 -carboxylic acid (ACC) synthase, the key enzyme in the pathway of ethylene biosynthesis.
The expression of most of the Aux/IAA family of early genes is stimulated by auxin within 5 to 60 minutes of hormone addition, and the induction is generally insensitive to cycloheximide. It is concluded that the members of the Aux/IAA gene family encode short-lived transcription factors that function as activators or repressors of the expression of the late auxin-inducible genes.
The members of the SAUR early gene family are probably related to tropic responses like phototropism’s and geotropisms, where auxin undergoes lateral transport.
In vertical seedlings, SAUR gene expression is symmetrically distributed. Within 20 minutes after the seedlings are placed horizontally, gravitropism leads to a rapid asymmetry in the accumulation of SAUR mRNAs, which begin to accumulate in the lower half of the hypocotyls.
This proves the existence of a lateral gradient in SAUR gene expression. The stimulation of SAUR genes by auxin is very rapid, occurring within 2 to 5 minutes of treatment. Likewise, the members of GH3 early gene family are also stimulated by auxin within 5 minutes. The function of the proteins encoded by such genes remains unknown.
It appears that among the major classes of early auxin-responsive genes, the first two, viz., the Aux/IAA and the SAUR genes are required for growth and development induced by auxin. On the other hand, GST-like genes and ACC synthase gene have their primary role in protection from stress damage.
(g) Auxin and Cell Cycle:
When tobacco cells are grown in culture medium with cytokinin but without auxin, the cell cycle is arrested at the end of either G1 or G2 phase, and eel division is stopped.
The cell cycle starts only after the addition of auxin. It is known that cyclin-dependent protein kinases (CDKs) together with their regulatory subunits, the cyclins, regulate the transition from G1 to S and from G2 to M (mitosis) during the cell cycle. Auxin exerts its effect on the cell cycle by stimulating the synthesis of CDKs and cyclins.
5. Bioassay Methods in Auxins:
There are a number of features which characterize an ideal bioassay method.
These are:
(i) High sensitivity
(ii) High specificity
(iii) Utilization of readily available material
(iv) Simplicity of execution and minimum requirement of special facilities and equipment
(v) Rapidity
(vi) Insensitivity to changes in environment and inhibitory substances
(vii) Dose/response relationship (or log dose/response or log dose/log response) which should be linear over a wide range of concentrations.
The bioassay methods which have been employed for the estimation of auxin in a plant tissue may be summarized as follows:
(a) Coleoptile Curvature Test:
The physiological basis for the test known as Went’s Avena coleoptile test lies in the polar basipetal transport of auxin in the organ. Increased concentrations of auxin contained in the agar blocks are applied on one side of decapitated coleoptiles obtained from dark-grown seedlings with occasional exposure to weak red light.
A difference in growth rate between the side of that coleoptile on which auxin is applied results in curvature of the coleoptile which is proportional to the amount of auxin applied. Auxin concentration in any tissue may be calculated by reference to the calibration curve obtained by plotting the angles of curvatures produced in response to known auxin concentrations.
(b) Coleoptile Section Straight Growth Test:
The coleoptile section straight growth test has been found to be simpler than the coleoptile curvature test, yet it is equally sensitive. Oat (Avena sativa) or wheat (Triticum aestivum) seeds are germinated and the seedlings are allowed to grow under weak red light.
Then segments of coleoptiles measuring 6 mm are cut 2 mm below the coleoptile tip. Batches of several such sections are incubated in small petri dishes containing different concentrations of auxin. At the end of the 24 h incubation period, a calibration is prepared with straight growth values in presence of different auxin levels from which the auxin content in an unknown sample can be determined.
(c) Split Pea Test:
About 6 cm of stem segments from the third internode of pea seedlings are taken after eight days’ growth. The segments are incised 3 cm down the middle of the stem and placed in test solutions in petri dishes.
Here auxin enters along the entire length of the stem and as the epidermal cells show greater growth in length than the inner cortical cells, the growth stimulated by auxin results in curvature response. Because of this differential growth on the two sides, the extent of curvature obtained with a solution to be tested is compared with a curvature produced by known auxin concentration.
(d) Oat Mesocotyl Straight Growth Test:
The method is the same as that of the coleoptile segment straight growth test. Here the seedlings are raised in complete darkness without any red light treatment to allow the growth of the mesocotyl.
The material is harvested when the mesocotyl is about 2.5 cm long (coleoptile length being 0.5) and 4 mm segments are cut just below the nodal zone. A measurable growth is obtained even with a low concentration of auxin and this test is as sensitive as the Avena curvature test.
(e) Pea Root Test:
The root cells are extremely sensitive to low concentrations of auxins and pea roots have been shown to yield quantitative promotion of growth in length in response to very low auxin concentrations. Straight, thin and uniform root segments are cut and placed in test solutions.
The growth obtained is roughly proportional to the logarithm of the concentrations of IAA and is thousand times more sensitive than other straight growth tests. The highest growth is generally obtained at 10-7M IAA and at higher concentration there is root growth inhibition.
(f) Root Inhibition Test:
Since auxin acts as a potent inhibitor of root elongation in intact seedlings, the inhibition of root growth by auxins is used as a quantitative test.
The simple technique consists of placing the young germinating seedlings on the rim of a cylinder of filter paper lined inside a glass tube containing the solution for testing. This allows straight growth of the root and the rate of elongation inhibition is proportional to the concentration of auxin.