Plant Biotech: Auxins

Nature of Auxins

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

Discovery of Auxins

Auxins were the first plant hormones determined. 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 outcomes of light on motion of canary grass (Phalaris canariensis) coleoptiles.

Darwin's try out advised that the tip of the coleoptile was the tissue contributing for perceiving the light and raising 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 encouraging the results of his first experiment (Darwin, 1880).

In 1913, Boysen-Jensen altered Fritting's experimentation by inserting parts of mica to block the transfer of the signal and showed that transport of auxin toward the base occurs on the dark side of the plant as contradicted to the side open to the unidirectional light (Boysen-Jensen, 1913). In 1918, Paal supported Boysen-Jensen's results by cutting off coleoptile tips in the dark, uncovering 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).

In 1926, a graduate student from Holland by the name of Fritz Went publicized a paper 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 (see below). 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.(see below)

Went called the hormone auxin (auxein: to grow). It took 20 years before this auxin was identified chemically as indole-3-acetic acid. Since then additional natural auxins have been identified.

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. 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).

Auxin, as plant hormone can be divided into two categories : Natural auxins and Synthetic auxins

Natural Auxins :

indole-3-acetic acid (IAA)
4-chloroindole-3-acetic acid (4-Cl-IAA)
phenylacetic acid (PAA)
indole-3-butyric acid (IBA

Synthetic Auxins :

2,4-Dichlorophenoxyacetic acid (2,4-D)
α-Naphthalene acetic acid (α-NAA)
2-Methoxy-3,6-dichlorobenzoic acid (dicamba)
4-Amino-3,5,6-trichloropicolinic acid (tordon or picloram)
2,4,5-Trichlorophenoxyacetic acid (2,4,5-T)

Effects of auxin

The major roles of auxin in tissue culture were established by Skoog and Miller in 1957. They observed that pith tissues excised from tobacco stems form shoots at high cytokinin and low auxin concentration, roots at low cytokinin and high auxin concentration, or callus at intermediate concentrations of both plant hormones. The formation or foots from pith fragments corresponds with the effect of auxin on rooting of cuttings, and the reduction of shoot formation with the inhibition of the outgrowth of axillary buds by auxin. A few years after the classical Skoog and Miller experiment, the formation of somatic embryos was observed after treatment with 2,4-D.

It should be noted that auxins are only required during the initial phases of adventitious root formation and somatic embryogenesis. After that, they become inhibitory and block the outgrowth of the root initials and embryos.

The effect of hormones is restricted bottom a specific period of time during the development and to specific tissues/cells. The rhizogenic action of auxins in apple microcuttings is 24h-96h after start of the rooting treatment and is restricted to specific cells near the interfascicular cambium adjacent to the vascular bundles.

2,4-D is often referred to as a strong auxin but this only applies to the formation of callus and somatic embryos : 2,4-D is a weak auxin with respect to the formation of adventitious root primordial or the inhibition of axillary buds. In contrast, IAA or IBA are not very effective in the formation of callus and somatic embryos, but show a high performance with the respect to adventitious root formation and inhibition of axillary buds.

Transport, uptake and Metabolism of Auxin

In plants, auxin is synthesized predominantly in the apical region and transported downwards. The underlying mechanism of this transport has been examined extensively. Uptake of auxin into cells occurs by diffusion and by active uptake via an influx carrier termed AUX1. The rate of uptake via diffusion depends on the dissociation of the molecule. Auxin in more protonated outside the plasmalemma than inside the cell (in the cell wall the pH is ca. 5.5 but the cytoplasm has a pH of ca. 7; IAA is a weak acid with a pKa of 4.7). The undissociated lipophilic auxin diffuses through the plasmalemma into the cell. In the cytoplasm the anionic form prevails, so auxin cannot easily diffuse out through the plasmalemma and is ‘trapped’ within the cells. Auxin is actively transported out of the cells bey efflux carriers, the PIN-proteins. Because the efflux carriers are located predominantly at the basal side of the cell, auxin is transported from the cell to cell in a basipetal direction, i.e., from apical to basal regions. Inside the cells, auxin moves from the apical to the basal side by diffusion. The rate of auxin transport is ca. one cm.h^-1. The active auxin transport occurs mainly in xylem parenchyma. Polarity itself is likely a major morphogenetic factor. In addition to directional transport, auxin can also move via water flow in the phloem.

When explants are cultured on medium with auxin, it is rapidly taken up probably via the same mechanism as described above (anion-trapping). This result in depletion of the medium. When plant tissues are cultured in liquid medium, most of the auxin may have disappeared from the mdium withi a few days. In solid medium only local exhaustion occurs because the slowness of diffusion over large distances. From the crucial medium components, auxin seems to be the only one that is so very rapidly depleted. The epidermis of plants is relatively impermeable to auxin and most uptake by explants occurs via the cut surface. How auxin reaches target tissues in the explants has not been studied. Roots are formed from founder cells close to the cut ends so auxin may reach these cells by diffusion.

Plant tissues inactive auxins by conjugation or enzymatic oxidation. All auxins can be conjugated. It is believed that conjugated auxin is inactive. However, conjugation is reversible and the free, active form may be released. It has been suggested that in the plants an equilibrium exists between the free and conjugated forms. Experimental data show that 2,4-D is slower conjugated than IAA, IBA or NAA. IAA is rapidly oxidized. MS-salts accelerate the rate of IAA oxidation. When using IAA, rapid photooxidation of IAA should be kept in mind. IAA is also unstable during autoclaving, but bioassays and chemical determinations show a loss less than 20%. IBA is slower photooxidized than IAA, whereas other auxins e.g. NAA, are not or only very little photooxidized. Riboflavin may be added to medium to enhance photooxidation of IBA. The photooxidation of IAA and of IBA in the presence of riboflavin may be turned to advantage. For example, in adventitious root formation cultures with IAA may be left in the dark unitl the root meristemoids have been formed by the rhizogenic action of auction (see figure). After that, when auxins have become inhibitory, the cultures are transferred to the light to degrade the auxin. It should be noted for the choice of auxin, chemical stability is only one of the factors to consider. The efficiency with respect to the developmental precess that should be promoted, is another major factor. The endogenous level of auxin and auxin action can be manipulated in various ways. In plant tissues, auxin is actively transported in a polar way. TIBA (triiodobenzoiz acid) and NPA (N-1-naphthylphthalamic acid) block this transport, because these compounds bind to the efflux carrier. The endogenous level of auxin can be increased by transforming plants with the auxin biosynthetic genes of Agrobacterium tumefaciens. The transformed plants show expected changes in their phenotype. Phenolic compounds (e.g., ferulic acid or phloroglucinol) may inhibit oxidation of applied auxin. This is not specific inhibition of enzymatic oxidation, photooxidation is also inhibited by adding phenolic compounds to the medium. PCIB is a genuine anti-auxin and competes with auxin for the auxin binding site at the auxin receptor.