Plant Physiology

Plant Physiology (Biology 327) - Dr. Stephen G. Saupe; College of St. Benedict/ St. John's University; Biology Department; Collegeville, MN 56321; (320) 363 - 2782; (320) 363 - 3202, fax; ssaupe@csbsju.edu

Plant Hormones - Auxin

I. Introduction
Auxin is a general name for a group of hormones that are involved with growth responses (i.e., elongate cells, stimulate cell division in callus). Not surprisingly, the term "auxin" is derived from the Greek word "to increase or grow". This was the first group of plant hormones discovered.

II. Chemistry/Structure

A. Naturally Occurring Auxins
The most important auxin found in plants is indole-3-acetic acid (IAA). IAA is comprised of an indole ring linked to acetic acid (see overhead). Other auxins that have been isolated from plants include indole ethanol, indole acetaldehyde, indole acetonitrile, phenylacetic acid (PAA), and 4-chloro-indoleacetic acid. These are probably converted to IAA in vivo.

B. Synthetics with Auxin Activity
There are a variety of substances that are not known to occur in plants that have auxin activity. These include indolebutyric acid (IBA); naphthalene acetic acid (NAA); 2,4- dichloro-phenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The exact mechanism of action of these compounds is not known, but they may inhibit nucleic acid synthesis.

C. Chemistry of action
Although a variety of molecular structures have auxin activity (i.e., derivatives of indole, benzene and naphthalene), these molecules seem to share a few features that appear to be required for activity. Auxin activity seems to be correlated with a flat, hydrophobic ring system that separates negatively-charged (acidic, carboxyl group) side chain and positively charged region. There is a charge separation of about 0.5 nm. Note that the indole unit isn't required for activity, but a planar ring system is.

D. Conjugated forms
Auxins, as do other hormones, occur in a free or conjugated (bound to sugars, alcohols or other molecules) form. In fact, up to 98% of the auxin may be bound. Auxins may be conjugated with inositol, coenzyme A or glucosides (sugars).

E. Anti-auxins
PCIB (p-chlorophenoxyisobutryic acid) is a compound that competes with auxin for binding sites. However, it doesn't cause any growth response.

III. Biosynthesis

A. Site
Auxin is made in actively growing tissue which includes young leaves, fruits, and especially the shoot apex. Made in cytosol of cells

B. Routes
There are two major routes to the production of IAA.

Tryptophan-dependent Pathways. The similarity of chemical structure of IAA and tryptophan suggested a connection between these. Considerable research has shown that tryptophan, one of the protein amino acids, is a precursor of auxin biosynthesis. Overall, the conversion of tryptophan to IAA can occur by: (1) a transamination followed by a decarboxylation; (2) a decarboxylation followed by a transamination; or (3) formation of IAA via an oxime (C=NOH) and nitrile (CN).
Tryptophan-independent Pathway - this route doesn't involve tryptophan directly as an intermediate to the formation of auxin.

IV. Transport

A. Basipetal (or Polar) Transport
Auxin is transported in a basipetal (towards the base, base-seeking) direction. In other words, auxin moves from the shoot tip towards the roots and from the root tip towards the shoot.

Evidence - (1) Seedling vs. [auxin]; and (2) ¹⁴C labeled IAA applied to the top of a stem section is recovered only from the bottom of the stem section. When auxin is applied to the end of stem segments, it is only transported from the "top" of the section to the "bottom" as demonstrated in these data:

	Upright	Upside down
¹⁴C-IAA in donor block (dpm)C-IAA in donor block (dpm)	10,000	10,000
¹⁴C-IAA in receiver block (dpm)C-IAA in receiver block (dpm)	7,500	300

B. Mechanism of polar transport

Transport Rate – IAA is not transported through the transpiration stream or phloem because the rate of movement is too slow. The rate of transport is consistent with diffusion.
Tissue of transport - appears to occur in parenchyma cells associated with the vascular tissue.
Model for polar transport: (a) Protons are moved out of the cell by a proton pump that requires ATP; (b) IAA is protonated at low pH; (c) IAA-H passes through the lipid membrane. It can enter or leave anywhere; (d) once inside the cell, the IAA-H ionizes in response to the higher pH; (e) IAA^- requires a permease to pass through the membrane; (f) histochemical studies have shown that a permease is only located at the bottom of the cell - resulting in a net movement of auxin out of the bottom of the cells.
Evidence for polar transport model: (a) transport is blocked by respiratory poisons (i.e., demonstrates the need for ATP and a proton pump); (b) unlabeled IAA competes with C¹⁴ labeled IAA for uptake in to the cells - this suggests that a carrier of some sort is required ; (c) fluorescein-labeled antibodies show that the permease is localized at bottom of cells; and (d) the transport of auxin is blocked by NPA (napthylthalamic acid), TIBA (tri-iodobenzoic acid) and flavanoids. These inhibitors appear to block the permease.
Proton-Auxin CoTransport Mechanism - In addition to the passive pH-dependent mechanism described, there a membrane transport protein seems involved that cotransports protons and IAA into the cell. This tranport protein is localized in the upper side of the cells.

V. Bioassays

There are four classic bioassays for auxin. These tests, which are all based on the ability of auxin to stimulate shoot growth (or inhibit root growth) are:

A. Avena coleoptile curvature test
Pioneered by F. Went. The angle of curvature of a decapitated oat coleoptile is measured after placing an agar block containing auxin on one side. Then, angle of curvature vs. [IAA] is plotted.

B. Avena coleoptile elongation
The ratio of final length/original length for oat coleoptiles or pea stem sections is measured after the tissues are floated in solutions containing different concentrations of IAA. Elongation vs. [IAA] is plotted.

C. Split pea curvature test
A section of pea hypocotyl is obtained and split halfway down the middle. After incubating the sections in solutions of known IAA concentration, the angle of curvature is measured. Note that only the epidermal cells are responding to the treatment.

D. Cress root inhibition
This bioassay is based on the ability of auxin to stop root growth. A ratio of treatment/control growth is plotted vs. log [IAA].

VI. Auxin responses
IAA is involved in the following responses:

A. Cell elongation and wall relaxation
    Discussed earlier in the semester. Basis of several bioassays and the discovery of auxin. NOTE: Normally exogenous application of IAA has little effect on plants.

B. Cell differentiation
    Promotes differentiation of vascular tissue (i.e., xylem & phloem).

C. Ethylene production
    IAA apparently stimulates the production of ethylene.

D. Inhibition of root growth
    [IAA] > 10^-6 M inhibit root elongation. However, very low (>10^-8 M) favor root elongation.

E. Stimulate root initiation (lateral roots, adventitious roots)
    Roots always form at the basal end of cutting

F. Flowering
    Although most plants don’t initiate the production of flowers after auxin treatment, pineapple and its relatives (Bromeliaceae) do. Once flowers are initiated, in many species, IAA promotes the formation of female flowers, especially in cucurbits (gourd family).

G. Parthenocarpic fruit development - as per RCBr Lab

H. Apical dominance
    The apical meristem (apex) controls or dominates the development of the lateral buds. (PS - a bud is an embryonic shoot with immature leaves and stem). Apical dominance also occurs in roots. It is responsible for the Xmas tree shape of many trees and prevents an individual from becoming too top-heavy.

Function: (a) maintain upward growth; (b) maximize light absorption; (c) prevent top-heaviness; and (d) provide mechanism to replace the apical bud if it is removed by grazing or damage.

Thimann & Skoog (1934) - first suggested a correlation between [IAA] and apical dominance. This idea was further developed by Cholodny-Went who proposed that plant tissues responded to different concentrations of IAA (see figure). For example, hi [IAA] stimulates shoot growth but inhibits bud and roots. Thus, IAA is produced at the tip of the plant and is transported downward. The high concentrations near the apex inhibit lateral buds. As the concentration decreases it frees the buds from the inhibition and they develop.

Evidence:

every gardener knows that removing the apical meristem results in bushier plants. This is consistent with the Cholodny-Went hypothesis. If IAA is applied to a de-tipped plant it prevents lateral bud development

IAA stimulates the formation of ethylene which is known to inhibit lateral bud formation.

branching mutants of tomato don't export ¹⁴C-IAA

But:

[IAA] in buds after the tip is removed should decrease, and they don't

[IAA] that act to replace the tip are much higher than levels in the intact plant
^{14C-IAA doesn’t enter the lateral buds when
applied at tip
Some plants don’t respond to exogenous IAA}

Nutrient diversion hypothesis
There is little development of vascular tissue to the lateral buds. This has lead some to propose that the lateral buds fail to develop because they get no nutrients because the nutritional traffic is being diverted to the apex.

Evidence: (a) application of cytokinins stimulates lateral bud development; cytokinins known to direct nutritional traffic, especially during senescence; (b) tomato mutants that exhibit strong apical dominance have lower levels of cytokinin; (c) there is a good correlation between cytokinin level and bud development.
Bud Inhibition
Another possible reason why buds fail to develop is because they have an inhibitor. It has been shown that ABA inhibits bud and seed development in many species. The buds of decapitated plants have a lower [ABA] than intact plants. High [ABA] in the bud are maintained by applying IAA to decapitated apex.

I. Tropic responses - such as gravitropism and phototropism

J. Abscission
Formation of the abscission layer is correlated with the [IAA] in leaf. As long as the [IAA] in the leaf is high relative to the stem, then the abscission layer doesn’t form. When the [IAA] in the leaf drops, which occurs normally during the growing season, it stimulates the formation of the abscission layer forms and the leaf falls. The IAA presumably works by decreasing the sensitivity of the cells to ethylene which is the primary hormone involved in initiating leaf drop.

VII. Regulating Endogenous Levels
All of the general methods (i.e., degradation, conjugates, regulate rate of synthesis, sequestering in different regions) of regulation are probably operative. In particular, IAA is probably shuffled back and forth between the two main areas, or pools, where it is stored – the cytosol and chloroplast. IAA oxidase, a type of peroxidase, converts IAA to inactive metabolites. There are several degradative pathways.