lect2 - Auxin 2005

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Nature of Auxins The term auxin is derived from the Greek word auxein which means to grow. Compounds are generally considered auxins if they can be characterized by their ability to induce cell elongation in stems and otherwise resemble indoleacetic acid (the first auxin isolated) in physiological activity. Auxins usually affect other processes in addition to cell elongation of stem cells but this characteristic is considered critical of all auxins and thus "helps" define the hormone (Arteca, 1996; Mauseth, 1991; Raven, 1992; Salisbury and Ross, 1992).

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History of Auxins and Pioneering Experiments Auxins were the first plant hormones discovered. Charles Darwin was among the first scientists to dabble in plant hormone research. In his book "The Power of Movement in Plants" presented in 1880, he first describes the effects of light on movement of canary grass (Phalaris canariensis) coleoptiles. The coleoptile is a specialized leaf originating from the first node which sheaths the epicotyl in the plants seedling stage protecting it until it emerges from the ground. When unidirectional light shines on the coleoptile, it bends in the direction of the light. If the tip of the coleoptile was covered with aluminum foil, no bending would occur towards the unidirectional light. However if the tip of the coleoptile was left uncovered but the portion just below the tip was covered, exposure to unidirectional light resulted in curvature toward the light. Darwin's experiment suggested that the tip of the coleoptile was the tissue responsible for perceiving the light and producing some signal which was transported to the lower part of the coleoptile where the physiological response of bending occurred. He then cut off the tip of the coleoptile and exposed the rest of the coleoptile to unidirectional light to see if curving occurred. Curvature did not occur confirming the results of his first experiment (Darwin, 1880).

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It was in 1885 that Salkowski discovered indole-3-acetic acid (IAA) in fermentation media (Salkowski, 1885). The isolation of the same product from plant tissues would not be found in plant tissues for almost 50 years. IAA is the major auxin involved in many of the physiological processes in plants (Arteca, 1996). In 1907, Fitting studied the effect of making incisions on either the light or dark side of the plant. His results were aimed at understanding if translocation of the signal occurred on a particular side of the plant but his results were inconclusive because the signal was capable of crossing or going around the incision (Fitting, 1907).

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In 1913, Boysen-Jensen modified Fritting's experiment by inserting pieces of mica to block the transport of the signal and showed that transport of auxin toward the base occurs on the dark side of the plant as opposed to the side exposed to the unidirectional light (Boysen-Jensen, 1913). In 1918, Paal confirmed Boysen-Jensen's results by cutting off coleoptile tips in the dark, exposing only the tips to the light, replacing the coleoptile tips on the plant but off centered to one side or the other. Results showed that whichever side was exposed to the coleoptile, curvature occurred toward the other side (Paal, 1918). Soding was the next scientist to extend auxin research by extending on Paal's idea. He showed that if tips were cut off there was a reduction in growth but if they were cut off and then replaced growth continued to occur (Soding, 1925).

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In 1926, a graduate student from Holland by the name of Fritz Went published a report describing how he isolated a plant growth substance by placing agar blocks under coleoptile tips for a period of time then removing them and placing them on decapitated Avena stems (Went, 1926). After placement of the agar, the stems resumed growth. In 1928, Went developed a method of quantifying this plant growth substance. His results suggested that the curvatures of stems were proportional to the amount of growth substance in the agar (Went, 1928). This test was called the avena curvature test. Much of our current knowledge of auxin was obtained from its applications. Went's work had a great influence in stimulating plant growth substance research.

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Above is the initial experiment described by Went (Arteca, 1996)

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Above is an illustration of Went's avena coleoptile test. Remove tip and place on agar for 1-4 hrs. b) Agar block containing the diffused chemical is then placed on one side of the coleoptile base. c) After 90-120 minutes, the angle of curvature is then measured. (Raven, 1992).

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He is often credited with dubbing the term auxin but it was actually Kogl and Haagen-Smit who purified the compound auxentriolic acid (auxin A) from human urine in 1931 (Kogl and Haagen-Smit, 1931). Later Kogl isolated other compounds from urine which were similar in structure and function to auxin A, one of which was indole-3 acetic acid (IAA) initially discovered by Salkowski in 1985. In 1954 a committee of plant physiologists was set up to characterize the group auxins. The term comes from the Greek auxein meaning "to grow." Compounds are generally considered auxins if they are synthesized by the plant and are substances which share similar activity to IAA (the first auxin to be isolated from plants) (Arteca, 1996; Davies, 1995).

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Biosynthesis and Metabolism of Auxin IAA is chemically similar to the amino acid tryptophan which is generally accepted to be the molecule from which IAA is derived. Three mechanisms have been suggested to explain this conversion: Tryptophan is converted to indolepyruvic acid through a transamination reaction. Indolepyruvic acid is then converted to indoleacetaldehyde by a decarboxylation reaction. The final step involves oxidation of indoleacetaldehyde resulting in indoleacetic acid. Tryptophan undergoes decarboxylation resulting in tryptamine. Tryptamine is then oxidized and deaminated to produce indoleacetaldehyde. This molecule is further oxidized to produce indoleacetic acid.

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As recently as 1991, this 3rd mechanism has evolved. IAA can be produced via a tryptophan-independent mechanism. This mechanism is poorly understood, but has been proven using trp(-) mutants. Other experiments have shown that, in some plants, this mechanism is actually the preferred mechanism of IAA biosynthesis.

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The enzymes responsible for the biosynthesis of IAA are most active in young tissues such as shoot apical meristems and growing leaves and fruits. The same tissues are the locations where the highest concentrations of IAA are found. One way plants can control the amount of IAA present in tissues at a particular time is by controlling the biosynthesis of the hormone. Another control mechanism involves the production of conjugates which are, in simple terms, molecules which resemble the hormone but are inactive. The formation of conjugates may be a mechanism of storing and transporting the active hormone. Conjugates can be formed from IAA via hydrolase enzymes. Conjugates can be rapidly activated by environmental stimuli signaling a quick hormonal response. Degradation of auxin is the final method of controlling auxin levels. This process also has two proposed mechanisms:

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The oxidation of IAA by oxygen resulting in the loss of the carboxyl group and 3-methyleneoxindole as the major breakdown product. IAA oxidase is the enzyme which catalyzes this activity. Conjugates of IAA and synthetic auxins such as 2,4-D can not be destroyed by this activity. C-2 of the heterocyclic ring may be oxidized resulting in oxindole-3-acetic acid. C-3 may be oxidized in addition to C-2 resulting in dioxindole-3-acetic acid. The mechanisms by which biosynthesis and degradation of auxin molecules occur are important to future agricultural applications. Information regarding auxin metabolism will most likely lead to genetic and chemical manipulation of endogenous hormone levels resulting in desirable growth and differentiation of important crop species. Ultimately, the possibility exists to regulate plant growth . without the use of hazardous herbicides and fertilizers (Davies, 1995; Salisbury and Ross, 1992).

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Functions of Auxin The following are some of the responses that auxin is known to cause (Davies, 1995; Mauseth, 1991; Raven, 1992; Salisbury and Ross, 1992). Stimulates cell elongation Stimulates cell division in the cambium and, in combination with cytokinins in tissue culture Stimulates differentiation of phloem and xylem Stimulates root initiation on stem cuttings and lateral root development in tissue culture Mediates the tropistic response of bending in response to gravity and light The auxin supply from the apical bud suppresses growth of lateral buds Delays leaf senescence Can inhibit or promote (via ethylene stimulation) leaf and fruit abscission Can induce fruit setting and growth in some plants Involved in assimilate movement toward auxin possibly by an effect on phloem transport Delays fruit ripening Promotes flowering in Bromeliads Stimulates growth of flower parts Promotes (via ethylene production) femaleness in dioecious flowers Stimulates the production of ethylene at high concentrations

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1. Phototropism Plant shoots display positive phototropism: when illuminated from one direction, the shoot proceeds to grow in that direction. Mechanism The direction of light is detected at the tip of the shoot. Blue light is most effective. It is absorbed by a flavoprotein called phototropin. Flavoproteins contain flavin as a prosthetic group. Auxin is synthesized at the tip and translocated down in the vascular tissue. Auxin transporters - called PIN proteins - are inserted in the plasma membrane of cells on the shady side of the shoot. Auxin is pumped out of these efflux transporters and stimulates elongation of the cells on the shady side causing the shoot to bend toward the light.

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2. Gravitropism Gravitropism is a plant growth response to gravity. Plant shoots display negative gravitropism: when placed on its side, a plant shoot will grow up Roots display positive gravitropism: they grow down. Possible Mechanism of Gravitropism in Roots When a root is placed on its side, Amyloplasts (organelles containing starch grains) settle by gravity to the bottom of cells in the root tip. The amyloplasts may be attached to actin filaments which are also attached to vesicles containing PIN proteins. When inserted in the plasma membrane, PIN proteins pump auxin out of the cell; that is, they are efflux transporters. Auxin sent down from the shoot arrives in the central tissues of the root tip. Tethered to amyloplasts, PIN proteins are inserted in the plasma membrane on the underside of pericycle cells causing auxin to accumulate on the under side of the root. This INHIBITS root cell elongation. [View reason for this.] So the cells at the top surface of the root elongate, causing the root to grow down.

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3. Apical dominance Growth of the shoot apex (terminal shoot) usually inhibits the development of the lateral buds on the stem beneath. This phenomenon is called apical dominance. If the terminal shoot of a plant is removed, the inhibition is lifted, and lateral buds begin growth. Gardeners exploit this principle by pruning the terminal shoot of ornamental shrubs, etc. The release of apical dominance enables lateral branches to develop and the plant becomes bushier. The process usually must be repeated because one or two laterals will eventually outstrip the others and reimpose apical dominance. Apical dominance seems to result from the downward transport of auxin produced in the apical meristem. In fact, if the apical meristem is removed and IAA applied to the stump, inhibition of the lateral buds is maintained.

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4. Fruit development Pollination of the flowers of angiosperms initiates the formation of seeds. As the seeds mature, they release auxin to the surrounding flower parts, which develop into the fruit that covers the seeds. Some commercial growers deliberately initiate fruit development by applying auxin to the flowers. Not only does this ensure that all the flowers will "set" fruit, but it also maximizes the likelihood that all the fruits will be ready for harvest at the same time.

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5. Abscission Auxin also plays a role in the abscission of leaves and fruits. Young leaves and fruits produce auxin and so long as they do so, they remain attached to the stem. When the level of auxin declines, a special layer of cells - the abscission layer -forms at the base of the petiole or fruit stalk. Soon the petiole or fruit stalk breaks free at this point and the leaf or fruit falls to the ground. Fruit growers often apply auxin sprays to cut down the loss of fruit from premature dropping.

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6. Root initiation Auxins stimulate the formation of adventitious roots in many species. Adventitious roots grow from stems or leaves rather than from the regular root system of the plant. Horticulturists may propagate desirable plants by cutting pieces of stem and placing them base down in moist soil. Eventually adventitious roots grow out at the base of the cutting. The process can often be hastened by treating the cuttings with a solution or powder containing a synthetic auxin.

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Translocation of Auxin Auxin travels from cell to cell being taken up on one side of the cell by influx transporters in the plasma membrane and moved out on the other side by efflux transporters - called PIN proteins - where it can then be taken up by the adjacent cell. So the distribution on the cell of these two types of transporter establishes which direction auxin travels in.

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How does auxin achieve its many different effects in the plant? Auxin effects are mediated by two different pathways: immediate, direct effects on the cell turning on of new patterns of gene expression 1. Direct effects of auxin The arrival of auxin at the surface of the cell initiates such immediate responses as changes in movement of ions in and out of the cell through the plasma membrane extension of the cell wall causing elongation of the cell. Auxin initiates these events after binding to specific receptors at the cell surface, probably transmembrane proteins such as ABP1 ("Auxin-binding protein 1")

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2. Effects of auxin on gene expression Many auxin effects are mediated by changes in the transcription of genes. The steps appear to be: auxin enters the cell by active transport through special auxin transporter molecules in the plasma membrane Auxin binds to molecules in the cytosol such as ARF1 ("auxin response factor 1") ARF1 is a transcription factor it enters the nucleus and binds to the DNA sequence TGTCTC ACAGAG This sequence is found in the promoters of auxin-responsive genes; that is, it is an auxin response element The action of auxin on gene transcription is quite similar to the action of steroid hormones in animals

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Synthetic auxins as weed killers Some of the most widely-used weed killers are synthetic auxins. These include 2,4-dichlorophenoxy acetic acid (2,4-D) and 2,4,5-trichlorophenoxy acetic acid (2,4,5-T). As the formulas show, 2,4,5-T is 2,4-D with a third chlorine atom, instead of a hydrogen atom, at the #5 position in the benzene ring (blue circles). 2,4-D and its many variants are popular because they are selective herbicides, killing broad-leaved plants but not grasses (no one knows the basis of this selectivity). Why should a synthetic auxin kill the plant? Auxin (IAA) is actively transported into cells by a transmembrane transporter and leaves the cells by facilitated diffusion through a different transporter. It turns out that the importer works fine for 2,4-D but that 2,4-D cannot leave the cell through the exporter. Perhaps it is the resulting accumulation of 2,4-D within the cell that kills it. A mixture of 2,4,-D and 2,4,5-T was the "agent orange" used by the U.S. military to defoliate the forest in parts of South Vietnam.

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