13.2—
Auxin Actions
13.2.1—
Introduction
Amongst plant growth regulators, the auxins have been studied the longest and have involved the greatest amount of research. This class of hormones is known to promote cell enlargement or cell elongation, a process which requires extension of the cell wall. Figure 13.1 shows the chemical structures of the major natural and synthetic auxins.
Figure 13.1
Structure of various auxins.
Commencing in 1940, the majority of studies involving auxins were begun in an attempt to determine the molecular interaction with the plant cell. Most of this research was directed toward the cell wall because it was believed that auxin
action required a change in the cell wall to allow for cellular expansion. Thus, the effects of the hormone were thought either to change the cell wall deposition or to hydrolyse certain cross-linkages in cell walls making them more elastic.
This type of research was carried out for several years without achieving a clear understanding of how auxin might control such processes within the cell wall. In the 1950's a new area of research was begun. Under the direction of F. K. Skoog in Madison, Wisconsin, a report (Silberger & Skoog, 1953) was published which showed that the auxin, indole-3-acetic acid, remarkedly affected the RNA and DNA contents of plants. Auxin increased the content of nucleic acids in tobacco tissue cultured on a sucrose-agar medium. This increase occurred prior to the auxin-induced growth of the tissue at concentrations of IAA which were optimal for cell enlargement. For many years following this discovery much research was devoted to the general mechanism by which auxin increased the synthesis of nucleic acids.
A great share of this research was begun in J. Hanson's laboratory in Urbana, Illinois by West and Key (West et al., 1960) who showed that the synthetic auxin, 2,4-D, (see (Fig. 13.1), produced a wide range of morphological and physiological changes in the hypocotyl of soybean and the mesocotyl of corn.
Within 15–24 hours after 2,4-D treatment, cellular enlargement was noted concomitant with an increase in the size of the nucleus. Accompanying these changes were dramatic increases in RNA content, most of which were due to increased ribosomal-RNA. Chrispeels and Hanson (1962) suggested that auxin acts on the nucleus causing it to revert to a meristematic type of metabolism. The role of the nucleus in such a sequence of events is of obvious importance since the nucleus has been shown to accumulate RNA in response to auxin. Beginning in the 1960's, Key began experiments on auxin regulation of nucleic acid synthesis. He and Ingle (1964) demonstrated that auxin controls the synthesis of nucleic acids other than that of ribosomal-RNA. Their data revealed that auxin causes production of nucleic acid which appears to be of the messenger-RNA type.
Subsequent experiments by O'Brien et al., (1968) showed that treatment of soybean with auxin caused a large increase in chromatin-directed RNA synthesis. It was of interest to note that auxin-induced RNA synthesis produced a type of RNA which was different from the control RNA as judged by molecular size and nearest neighbour analysis. Following this area of research other studies (Hardin & Cherry, 1972; Hardin et al., 1970; Hardin et al., 1972) showed that 2,4-D increased the activity of RNA polymerase. Current research suggests that particular cytoplasmic or membrane bound factors may enhance the activity of RNA polymerase. It is hypothesized (Hardin et al., 1972) that the mechanism of action of auxin is to bind to a receptor molecule located on the plasma membrane. The receptor is then released and moves into the nucleus where it modulates the activity of the RNA polymerase. The increase in RNA polymerase activity leads to increased synthesis of messenger-RNAs, which in turn regulate, or control, the synthesis of specific proteins within the target cells.
A primary challenge is to isolate the factor which binds to, or reacts with, auxin, and determine whether this is the primary action of the hormone. Secondarily, research is needed to determine what series of events this interaction puts into metabolic play. It is likely, once the auxin has bound to the receptor molecule within the target cells, that the changes in nucleic acid synthesis cause an increased activity of the various enzymes associated with the cell wall. Many other activities which have been measured over the many years are probably secondary effects resulting from the primary action of the auxin reacting with its receptor molecule.
13.2.2—
Regulation of Cell Wall Extension Ability
When plant cell walls elongate, an accompanying increase in weight and volume also takes place. This increase in size requires that the cell wall be increased in mass as well as area. Even though the dry weight of wall material greatly increases due to cellular enlargement, the thickness and density of the wall remains constant. Thus, wall synthesis appears to be a fundamental requirement during cell elongation. In addition, an increase in wall mass must come from a deposition of polysaccharides. It was believed for many years that auxin, in this particular case indoleacetic acid, increased cell elongation by promoting cell wall synthesis. Auxin, in order to allow for the increased wall extensibility, may also regulate the activity of enzymes involved in cell wall loosening which catalyse the breaking of various cross-linkages between the wall microfibrils.
Plant tissues which respond to auxin by an increased rate of cellular elongation also exhibit an increase in cell wall loosening. However, the kinetics of auxin-induced wall loosening vary considerably from tissue to tissue. For example, in dark grown maize coleoptiles wall loosening is induced by concentrations of auxin around 10–4M , and the increase in loosening proceeds very slowly with time. However, the same tissue responds to auxin very dramatically as maximal increases in total length occur between two and six hours after exposure to auxin. It is of interest, therefore, that the rate of cell wall elongation, as measured by increased section length, is much greater than the increase in cell wall extensibility.
Thus, considerable changes in cell wall extensibility in a given tissue as caused by auxin, may affect growth in a different manner. In certain tissues the rate of growth is constant whilst total wall extensibility changes. In other tissues wall extensibility remains constant during a period of changing growth rate. At the present time it seems that auxin causes a biochemical change in the wall probably by breaking or modifying the cross-links between the wall polysaccharide chains. These changes in the cell wall are then translated under cell turgor pressure into wall elongation. It is now relevant to discuss how auxin affects the cross-links in a cell wall and what the biochemical changes are which take place in a cell wall during cell elongation. In this regard, it is necessary to look at the effect of auxin on various enzymes associated with the cell wall in the context of how these enzymes affect cell wall loosening properties.
13.2.3—
Action on Cell Wall Associated Enzymes
The most dramatic auxin effect on enzyme formation is the induction of cellulase, polygalacturonase and other hydrolases in pea cotyledons treated with a high concentration of IAA (Datko & Maclachlan, 1968). Detection of in vitro formation of cellulase by a ribosomal preparation from peas has been claimed. This was reported to be enhanced using a ribosomal system prepared from IAA-treated peas (Davis & Maclachlan, 1969). The large IAA effect on cellulase formation in vivo is a slow response occurring over several days and cannot be involved in a rapid auxin action on elongation. However, it could be a cause of the lateral swelling response seen in pea cells following IAA treatment.
Auxin stimulates not only the synthesis of cellulase but also the level of glucan synthetase. Masuda (1968) and Masuda and Yamamoto (1970) found that the fungal b -1,3-glucanase, isolated from cultures of Sclerotinia libertans induced rapid elongation of excised oat coleoptile segments. This enzyme was shown to increase cell wall extensibility as measured by a stretching method. In other studies, Masuda et al. (1970), compared the activity of the endo-b -1,3-glucanase and its activity on Avena coleoptile cells with those of the exoenzyme. They found that exoglucanase enhanced elongation and extensibility of the cell wall. But the effect was not additive to the effect of IAA. Furthermore, at least three hours of incubation with exoglucanase was required for enhancement of elongation. The endoglucanase showed no effect on cell wall elongation. Cell wall turnover and auxin effects thereon in pea stem tissue have been studied using pulse-chase wall labelling experiments. Considerable turnover of galactan occurs but this is not influenced by IAA (Labavitch & Ray, 1974).
The action of auxin on soluble xyloglucan can be assayed with relative ease and precision and has been positively demonstrated to be in progress within thirty minutes after exposure to IAA and probably within fifteen minutes, placing it amongst the most rapid metabolic effects of auxin. This auxin effect is blocked by metabolic inhibitors that are known to block elongation, but persists under complete osmotic inhibition of elongation by mannitol. It seems likely that it is involved in the action of the cell wall that leads to elongation in pea cells. Involvement of xyloglucan is understandable in terms of a recent model for cell wall structure, according to which xyloglucan serves to bind matrix polysaccharides to cellulose microfibrils (Bauer et al., 1973; see also chapter 1).
13.2.4—
Hydrogen-Ion Pump
Since the 1930'S it had been known that acidic media can stimulate elongation in auxin-sensitive tissue. Recent work showed that acidic buffers or CO2 solutions in the pH range from 3 to 4 induce a rate of coleoptile cell elongation as great as or greater than that obtainable with auxin (Rayle & Cleland, 1970; Hager et al., 1971; Evans et al., 1971). Acid-induced elongation starts almost immediately upon treatment rather than after the 10–15 minute latent period
characteristic of auxin action. Acid-induced elongation is not suppressible by metabolic inhibitors, such as cyanide, mercurials, and cycloheximide, or by lack of oxygen, all of which block auxin-induced growth, and appears to be a passive process independent of metabolism. In later work it was found that when the epidermis is removed to improve H+ entry into the tissue, the full acid-pH stimulation of elongation develops over the pH range from about 6.0 to about 5.0 (Rayle, 1973; Cleland, 1973).
Rayle et al., (1970b) found that cell wall skeletons of frozen-thawed, dead coleoptile tissue would elongate dramatically if treated with acidic media while being held under tension by an applied load. Rayle and Cleland (1972) concluded that acid pH- and IAA-induced elongation must occur by the same mechanism, because the two kinds of elongation had similar rate, similar temperature dependences, and a similar yield threshold. While suggestive, these similarities do not establish identity of biochemical mechanism.
Hager et al., (1971) also felt, on grounds that IAA-induced elongation could be suppressed by alkaline media, that IAA-induced growth involves the same biochemical mechanism as acid-induced elongation. In support of their proposed auxin-stimulated H+ pump they offered experiments showing rapid stimulation of coleoptile elongation by ATP, ITP, and GTP under anaerobic conditions, although these results may have represented merely the acid pH effect itself, since these compounds are strong acids and were applied in unbuffered, pH 5 media.
In recent work an auxin-induced release of H+ ions from coleoptiles stripped of their epidermis (Cleland, 1973; Rayle, 1973) and from other auxin-sensitive tissues (Marrè et al., 1973; Ilan, 1973) has been detected. This is measured simply as a gradual fall in pH of the medium bathing the tissue, upon treatment with IAA, from near-neutral pH to pH 5 or below, i.e., into the range that by itself induces elongation. Detectable H+ secretion by coleoptiles begins within 20 to 30 minutes after exposure to IAA and may be regarded as a 'rapid response'. To the extent so far studied, H+ secretion by coleoptiles has a specificity for auxin analogues and antagonists similar to the specificity seen in growth, and a similar dependence on metabolism. Secretion is sensitive to inhibitors and uncouplers of energy metabolism and, perhaps unexpectedly for a transport process but just like cell enlargement, H+ secretion is quickly inhibited by low concentrations of cycloheximide (Rayle, 1973; Cleland, 1973). Comparable results with pea stem segments have been reported by Marrè et al., (1973).
These findings, coupled with the observations that induction of elongation by auxin is prevented by sufficiently well-buffered media of pH 6 to 8 provided the cuticle is removed or rendered permeable by gentle abrasion (Rayle, 1973), constitute presumptive evidence that the auxin effect on cell enlargement is mediated by externally secreted H+ ions.
The simplicity and directness of the acid secretion theory of auxin action is appealing and the theory has quickly become popular. Various auxin effects on cation and anion transport noted previously were considered by their authors
to be consistent with the H+ secretion theory. H+ secretion could involve either a parallel flow of anions, or a counterflow of, or exchange with, cations as has been inferred for H+ pumping by beet cells (Poole, 1973). However, as yet no immediate dependence on external ions of either auxin-induced H+ secretion or growth has been found.
Cleland (1973) and Rayle (1973) observed that auxin-treated coleoptiles cease to release H+ when the medium reaches pH 5.0 or slightly below, suggesting that auxin-stimulated H+ secretion is inhibited by acid pH, like other H+ pumps (Poole, 1973), or alternatively that, at pH 5, back-diffusion of H+ into the cell offsets the action of the pump. This feature should allow extensionpromoting pH values in the cell wall to be reached rapidly under auxin treatment, while shutting down further acidification which would probably be injurious to the cell (Cleland, 1973).
Some objections to the H+ secretion theory of auxin action and weaknesses in the evidence for it should be mentioned. For example, it has not yet been shown that suppression of auxin elongation by neutral buffers is not due to inhibited uptake of auxin, or to failure of primary action (the auxin binding found by Hertel et al., (1972) had a pH optimum below pH 6.5).
The most important piece of evidence still needed for the acid secretion theory is that the pH in the tissue's free space falls to an extension-stimulating value at the time that rapid auxin-induced elongation commences, i.e., within 10–15 minutes after exposure to auxin, as against the hour or more that is required for an external bathing medium to reach pH 5.0 to 5.5 (Cleland, 1973; Rayle, 1973). At the very least it must be demonstrated that IAA induces H+ secretion as quickly as it induces rapid elongation. Existing data fail this test and show an increase in H+ efflux from coleoptiles beginning, at the earliest, 20 or 30 minutes after exposure to IAA (Cleland, 1973; Rayle, 1973). These authors feel that the discrepancy may be attributed to time lags imposed by the free space volume and the diffusion path length for H+ to reach the external medium. A worse timing discrepancy was seen with sunflower hypocotyl (Ilan, 1973; cf. Uhrström, 1969). A more sensitive method for measuring H+ efflux needs to be employed to resolve these discrepancies. Better still, the pH within the free space of the tissue should be measured directly during response to auxin treatment.
13.2.5—
Regulation of Genetic Material
After the discovery of messenger-RNA it was suggested that the regulation of gene action by auxin required the specific control of messenger-RNA synthesis. Using a series of inhibitors, Key (1964) and Key and Ingle (1964) noted that treatment of soybean hypocotyl with 5-fluorouracil (5-FU) inhibited total RNA synthesis by 80% whilst growth proceeded normally. Further studies on the fractionation of nucleic acid from soybean hypocotyls on a methylated albumin Kieselguhr column showed that 5-FU inhibited the incorporation of labelled
adenosine diphosphate into ribosomal-RNA but had little effect on the labelling of messenger-RNA. Additional experiments with soybeans showed that 5-FU did not affect the tenaciously bound RNA whose synthesis is promoted by 2,4-D.
Sen and his colleagues (Roychoudury & Sen, 1964; Roychoudury et al., 1965) showed that nuclei isolated from coconut milk responded to auxin by making more RNA in vitro. On the basis of these observations, it was proposed that auxin directly affected the nucleus and thereby regulated gene expression. It was thought that in the presence of the hormone a greater amount of DNA template was exposed and allowed to be transcribed into RNA. In an attempt to test this hypothesis, Cherry (1967) showed that nuclei isolated from peanut cotyledons did not respond in vitro to 2,4-D at physiological concentrations (10–8 to 10–6 M ). From many other experiments, Cherry demonstrated that only in approximately 1 out of 10 attempts could nuclei be shown to respond to the hormone in vitro by the synthesis of more RNA. In general, absolutely no effect of the auxin on the capacity of the isolated nuclei to produce RNA was found. On the other hand, it was found routinely that isolated nuclei from soybean seedlings pretreated with 2,4-D produced twice as much nucleic acid as did control nuclei. Furthermore, experiments of O'Brien et al., (1968) showed that chromatin isolated from soybean hypocotyls did not respond in vitro to 2,4-D. In all cases the tissues needed to be treated with the growth regulator for at least two hours before any effect on chromatin-directed RNA synthesis could be noted. It is to be noted, however, that in a few experiments an effect of the hormone could be observed within thirty minutes. Subsequently, a progressive increase in chromatin-directed RNA synthesis was noted as a function of time. From those experiments, it was suggested that the auxin-enhancement was a result either of a more active RNA polymerase or of gene derepression leading to an increased availability of DNA template. A third possibility was that both of these effects were involved. Subsequent experiments demonstrated that in the presence of E. coli RNA polymerase, chromatin from both control and 2,4-D treated plants allowed the synthesis of similar amounts of RNA at saturation with the polymerase. Even though the total E. coli RNA polymerase activity with chromatin from 2,4-D treated plants was slightly higher than that with control chromatin, it was concluded that the auxin primarily promoted the endogenous RNA polymerase, rather than the chromatin template availability.
When it became possible to solubilize RNA polymerase from chromatin (Hardin & Cherry, 1972) two important aspects were noted. First of all, it has not been possible to show that the addition of auxin in vitro to solubilized RNA polymerase, or to chromatin, increases the rate of RNA synthesis (Hardin et al., 1970). Secondly, of the many experiments that have been performed, it appears that treatment of sensitive plants with auxin leads to a greater production of RNA polymerase I (Hardin & Cherry, 1972; Hardin et al., 1972). This is the enzyme which is thought to be present in the nucleoli, as judged from comparisons with animal RNA polymerase, and is the enzyme thought to transcribe ribosomal into ribosomal-RNA. These data agree with the fact that large increases
in ribosomal-RNA and ribosomes are observed after auxin treatment. However, they are inconsistent with the idea that auxin increases the activity of the RNA polymerase enzyme which transcribes unique DNA sequences into messenger-RNA.
13.2.6—
Action on Membranes
A current, interesting view of auxin action is that the hormone rapidly effects its action at, or on, cellular membranes, possibly involving the regulation of the export of growth active materials across the plasma membrane into the cell wall space. Auxin action at the plasma membrane was hypothesized by Hertel and Flory (1968) and by Rayle et al. (1970) partly in the belief that auxin transport and auxin action on growth involve closely related, if not identical, interactions, presumably with some carrier site located in the plasma membrane. Evidence for auxin action at the plasma membrane comes from observed IAA effects on membrane potentials (Tanada, 1970). This effect, however, has not yet been adequately studied nor shown to be related to the elongation process. Another indication of auxin action at the plasma membrane is the induction of cell elongation by certain fungal toxins which are known to increase membrane permeability (Evans, 1973).
It is assumed that specific auxin receptor sites must be located in or on the plasma membrane if the hormone is to change the permeability or to change any physical property within the membrane. With this general assumption in mind, Lembi et al. (1971) began to look at the binding of labelled NPA (naphthylphthallamic acid) a powerful competitive inhibitor of polar auxin transport which was considered to be likely to bind to specific auxin binding sites. Using maize particle preparations, they tentatively showed that there was a greater amount of NPA binding to fractions rich in plasma membranes in comparison to all other fractions obtained. IAA did not compete with NPA for the sites which thus do not seem to be the actual carrier sites for polar transport of IAA. It is of course conceivable that the sites binding NPA are not the same sites which would normally bind the auxin, IAA.
Hertel et al. (1972) detected a specific binding of labelled IAA and naphthaleneacetic acid (NAA) to certain particles in maize homogenates. Specific binding was assayed as radioactivity retained by particles exposed to 2 to 10 × 10–7 M labelled auxin minus radioactivity retained when, in addition, 10–4M unlabelled IAA or NAA was added to compete with and displace labelled auxin from saturateable binding sites. The radioactivity which remained in the latter case then was termed unspecific binding. This difference-assay gave specific binding with binding constants in the rage of 10– ; to 10–5M . Specificity of the binding as an auxin phenomenon was indicated by the evidence that analogues of IAA or NAA which are active on growth, and are transportable, can displace labelled IAA or NAA from the binding sites, whereas chemically similar, but biologically inactive, molecules do not compete in the binding assay.
One defect of these data was that 2,4-D, a very active auxin, competed relatively weakly with IAA or NAA in the assay, nor was specific binding of labelled 2,4-D itself detected. Based on the evidence that auxin-specific binding sites are localized in plasma membranes, Hardin et al. (1972) proposed that the reaction of auxin, either the native IAA or the synthetic 2,4-D, with the plasma membrane would probably lead to conformational changes within the membrane, causing the release of a receptor molecule into the cytoplasm. This receptor molecule may or may not be the same binding agent with which the auxin interacts. Nevertheless, the receptor released from the plasma membrane could then travel through the cytoplasm and into the nucleus where we know auxin ultimately increases the activity of RNA polymerase.
From previously published information from animal systems, (Shyamala & Gorski, 1969; Jensen et al., 1968) a good analogy can be drawn with hormone interaction with receptor molecules which then migrate into the nucleus where nucleic acid synthesis is controlled. Following these lines of investigation, Hardin et al. (1972) isolated a plasma membrane-rich fraction in which the plasma membrane was thought to be 70% homogeneous. The addition of plasma membranes to soybean RNA polymerase caused an increase in RNA synthesis in vitro. Subsequently, it was shown that pretreatment of the membranes with 10–7M 2,4-D greatly increased RNA polymerase activity. Still further, when the plasma membranes were pretreated with 2,4-D followed by removal by centrifugation, the supernatant fraction contained the RNA polymerase stimulus. The chemical release of this factor appears to be specific for auxin, i.e., IAA and 2,4-D bring about this release, but a non-auxin such as 2,5-D is totally inactive.
In other studies an attempt was made to determine which of the multiple RNA polymerases were stimulated by the auxin-released factor. It was found that the addition of plasma membrane factor to a semi-purified RNA polymerase preparation enhanced the a -amanitin sensitive polymerase. By analogy with animal RNA-polymerase studies, these data imply that auxin increases the nucleoplasmic RNA poly merase, the enzyme which is thought to transcribe DNA into messenger-RNA. If this is true then it is sensible to believe that auxin, through the release of a receptor molecule, may regulate the transcription of unique DNA sequences through the control of a specific enzyme.
13.2.7—
Summary
Because of rapid auxin responses on plant cells, over the last few years a whole new field of research has begun which relates not only to the gene concept but also to rapid responses. Therefore, the new area of research has dealt with actions of auxins on receptors, membranes and other binding surfaces which could lead to a rapid growth response, as well as a continued and long response involving nucleic acid synthesis and protein synthesis.
The apparent binding of auxin to plasma membrane sites has stimulated interest in the concept that the hormone might bring about conformational
changes within the membrane structure. This in turn might lead to changes in permeability, causing an increased ion flux across the membrane. If a receptor molecule is located on the plasma membrane, a change in conformation of the membrane might bring about a release of the receptor. It has been proposed that this receptor moves into the nucleus of the target cell and once there, increases the activity of RNA polymerase. This, in turn, leads to the synthesis of messenger-RNA which codes for proteins and this brings about the net result of auxin increased growth.
In this particular section the effects of cyclic-AMP on auxin controlled growth have not been covered. When cyclic-AMP was first found to stimulate plant growth, considerable excitement was generated (Solomon & Mascarenhas, 1971). However, that excitement was short-lived, and it now appears that cyclic-AMP has little or no effect on plant cells (Ownby et al., 1975).
A number of enzymes have been shown to be affected by auxin. These include the hydrolases, particularly cellulase, and other enzymes associated with cell wall degradation as well as cell wall synthesis. Whilst these enzymes are affected by the auxins, it is very likely that this result is a secondary effect of the auxin and is not related to the primary mechanism of action. In essence then, it appears that auxins control a multitude of physiological and biochemical responses in plants. It is possible that the primary site of action is localized within the plasma membrane or some surface binding site of the cell. A change at this particular level, either in structure or function, could bring about the release of a factor which moves into the nucleus. As a secondary amplification, the hormone thus modulates the activity of RNA polymerase. This in turn leads to the synthesis
Figure 13.2
A hypothetical model of auxin action. The interaction of auxin with the
plasma membrane results in the release of a factor which moves through
the cytoplasm and into the nucleus. The factor controls the activity of RNA
polymerase II in the nuclei which leads to synthesis of mRNA. The new species
of mRNA translated in the cytoplasm into new proteins leads to enhanced cellular growth.
of RNA from parts of the genome which were either not transcribed at all, or just to a low level in the absence of the receptor. A change in DNA transcription in this manner would lead to the synthesis of new proteins which would bring about the change in the growth potential (Fig. 13.2).