AUXINS

 

Plant Hormones

Plant hormones are naturally occurring organic molecules that regulate physiological processes. It is unknown how many plant hormones exist. They direct cellular differentiation, growth and developement. Tissues that are tageted by plant hormone react depending on location of  tissues and physio-chemical environment of tissues. A number of synthetic plant hormones have uses in grafting, tissue culture and plant propagation from cuttings. See http://www.tutorvista.com/content/biology/biology-iv/plant-growth-movements/synthetic-plant-hormones.php.

Auxins are a class of plant hormone, or phytohormone, produced in the tips of the stem and have an intergral part in many stages of the plant life-cycle. Four naturally occurring auxins have been discovered, the dominant is IAA auxin, a UV light sensitive phytochrome, which has the biggest impact on growth and development of roots and shoots. See also http://www.ncbi.nlm.nih.gov/pmc/articles/PMC438214/ for growth factors other than auxin. The presence of auxins in the plants circulatory system typically act to oppose or work with other hormones. Genes code for specialized PIN protens which regulate assymetrical auxin distribution in many stages of  plant development.

See www.annualreviews.org/doi/pdf/10.../annurev.cellbio.23.090506.123214

for more information on auxin receptors and plant development. For information of the importance of phytochrome-mediated phototropism, see http://www.plantphysiol.org/cgi/content/abstract/100/1/170.



'Auxin effect on root and shoot'



• Auxin induces cell elongation in shoots.
• Auxin inhibits cell elongation in roots

IAA auxin causes cell elongation in response to an external stimuli. See www.public.iastate.edu/~bot.512/lectures/Auxin-action.htm for the transport mechanisms of auxin. External environmental stimulents include gravity (geotropism), light (phototropism), and water (hydrotorpism). An unequal distribution of auxin produces cells with high IAA auxin concentrations and cells with low IAA auxin concentrations. In phototropism the high IAA auxin concentration cells are on the oppisite side of the light source.  






'Auxin distribution during phototropic response'

 

The root tissues and shoot tissues contain photorecepors that absorb light. It may be a positive or negative effect depending on the wavelength absorbed. Quantity, quality (wavelength) and direction of light can be detected by photoreceptors as well as several plant pigments, for exmple, photosynthetic pigments. The photorecepors, or phytochromes, of roots and shoots are positively reactive with red light, while blue light reacts positive with shoot photoreceptors and negative with root photoreceptors. Red light falls between 620-750nm, blue light falls between 450-490nm. Two forms of red light absorbing photoreceptors are Pr and Pfr.

'Light spectrum and wavelength measurements (nanometers)'



Roots grow down, showing positive gravitropism. Shoots grow upward, showing negative gravitropism. A build up of specialized cell organelles on the under side of roots inhibit cell elongation. The upperside of the roots elongate, directing the root downwards.

'Auxin distribution during geotropic response' 



See www.biology-online.org/3/5_plant_hormones.htm, for hormones and the effects of geo- and phototropism.

Root cap cells detect the presence of water and grow towards the higher gradient concentration. Since soil-water does not remain constant, it is difficult to show that water attracts elongating roots. Hydrotropism is mainly a phonomena observed in the laboratory.





'Hydrotropic response of root'



Auxin interacts with many plant systems or processe, such as;

• axial elongation of shoot tissue
• promotes root initiation and lateral expansion of root tissue
• inhibits the release of stress hormones and retards abscission. See http://www.jstor.org/stable/2438666 for further information.
• formation of transport system in vascular plants
• induces cell proliferation and differentiatiton to repair damaged vascular tissue
• induce flowering and retard fruit ripening 

EXPERIMENT: An investigation into the effect of Auxin (IAA) growth regulator on plant tissues

Materials.

• Distelled water
• Thermometer
• Beaker
• Radish seeds (Raphanus sativus)
• IAA solution (0.01% w/v)*
• Absorbent cotton woll
• Adhesive tape
• Incubator (25 degrees celsius)
• 8 Small graduated bottles
• 8 Petri dishes
• 8 Circular acetate grids
• 8 Pieces of filter paper

*Note: IAA is insoluble in water. To make a 0.001% solution, weigh out 0.1g of IAA powder and place in beaker. Add 2 ml of ethanol and stir to dissolve. Add 800 ml of distelled water and heat to 80 degrees celsius for 5 minutes, this evapourates the ethanol. After cooling to room temperature place in a volumetric flask and make up to 1litre with distelled water.

Procedure.

• Label petri dishes and graduated bottles as follows: 10^2 parts per million (ppm), 10 ppm, 1 ppm, 10^-1 ppm, 10^-2 ppm,10^-3 ppm, 10^-4 ppm, and one control with distelled water. The symbol ^ denotes 'to the power of'.
• Add 10 ml of 0.01% w/v IAA solution to the first graduated bottle.
• Add 9 ml of distelled water to the next 7 graduated bottles.
• With a Pasteur pipette, remove 1 ml of IAA solution from the first graduated bottle and add it to the second graduated bottle. Replace cap and mix well.
• With a new Pasteur pipette, remove 1 ml from the solution in the second graduated bottle and add it to the third graduated bottle. Replace cap and mix well.
• Repeat this serial dilution for the 4th, 5th, 6th and 7th graduated bottles, using a clean Pasteur pipette each time.



'Serial dilution'

• Removing 1 ml of soultion from the 7th graduated bottle gives all graduated bottles 9 ml of solution.
• Fit a circular acetate grid in the lid of each petri dish and place 5 radish seeds along one grid line. As shown in figure A.



Fig. A.  'Layout of radish seeds on acetate grid' 

• Place filter paper over the seeds in each petri dish.
• With a clean Pasteur pipette remove 2 ml from each graduated bottle and add it to its corresponding  IAA concentration petri dish e.g. 10^2 labelled graduated bottle to 10^2 labelled petri dish.
• press gently to remove any trapped air.
• Place absorbent cotton wool over the petri dish. The wool should be the appropiate size of the dish and about 0.5cm thick.
• Pour the remaining 7 ml of IAA solution into each corresponding petri dish and allow the cotton wool to absorb all IAA solution.
• Put the base on each petri dish and secure with adhesive tape.
• Place the dishes on their edge to ensure downward growth of roots.
• Incubate for 2-3 days at 25 degrees celsius.
• After incubation, measure the lenght of the roots and shoots of radish seedlings using the acetate grid
• Calculate the total length and the average length of roots and shoots. Results were recorded in tables 1a. and 1b.
• Estimate percentage stimulation or inhibition of root and shoot growth in each petri dish using the formula;                                      

              % stimulation = (average length – average length of control) × 100

                                                           average length of control

• Plot a graph of percentage stimulation and inhibition of root and shoot growth againt IAA concentration. (IAA should be on the y-axis)

 

Results.

 Table 1a: Root lengths of radish seedlings grown in different concentrations of IAA

Concentration of IAA (ppm)	Length of roots (mm)					Total length (mm)	Average length (mm)	% stimulation or inhibition
	Seed 1	Seed 2	Seed 3	Seed 4	Seed 5
0	67	36	68	0	0	171	57	0
10-4	9	7	4	6	0	26	6.5	-88.6
10-3	71	7	4	0	0	82	27.3	-52.1
10-2	50	75	106	32	4	267	53.4	-6.3
10-1	44	102	27	0	0	173	57.6	1.16
1	0	43	5	96	0	144	48	-15.8
10	25	57	56	23	0	161	40.25	-29.4
102	0	0	0	0	0	0	0	-100

               


 Table 1b: Shoot lengths of radish seedlings grown in different concentrations of IAA

Concentration of IAA (ppm)	Length of shoots (mm)					Total length (mm)	Average length (mm)	% stimulation or inhibition
	Seed 1	Seed 2	Seed 3	Seed 4	Seed 5
0	16	40	21	5	0	82	20.5	0
10-4	0	5	3	2	0	10	3.3	-83.8
10-3	37	3	0	0	0	40	20	-2.4
10-2	31	35	41	0	11	118	23.6	43.9
10-1	26	51	32	7	0	116	29	41.5
1	0	42	4	44	0	90	30	46.3
10	15	26	29	19	0	89	22.25	8.5
102	0	0	0	0	0	0	0	-100

See  www.curriculumonline.ie › ... › Prescribed Activities  for similar approach to procedure, as well as note taking and laboratroy safety precautions.



THE SCIENCE OF AUXINS ACTICITY INTRA- AND EXTRACELLULARY. 

Auxin induces the expression of a specific family of genes that translate mRNA into polypeptides like transport proteins, enzymes to synthesis structural componants of the cell wall, or Aux/IAA repressors for proeolysis of damaged or excess protein. Auxins consist of an aromatic ring with a carboxyl acid side chain (Fig. 1). Indole-3-acetic acid (IAA) biosynthesis occurs in the cytosol of growing tissues, predominantly in shoot apex and meristematic regions, and may follow one of two pathways. A tryptophan-independent pathway does not require tryptophan (Fig. 2) auxin synthesis. A tryptophan-dependent pathway uses tryptophan as a precursor to IAA. Decarboxylation and transamination of tryptophan takes place, with either of the two biochemial process taking place first followed by the other. The newly synthesised IAA molecules move down the stem in a unidirectionly manner. Auxin enters the cell at the apical end through the plasma membrane via passive transport, no is ATP used. It exits via active transport in which ATP is used. Cell wall (pH~5) and cytoplasm (pH ~7) have an proton gradient which allows IAA to pick up a hyrdogn ion, thus becoming electrically neutral (IAAH), and move through the plasma membrane. Ionization of  IAAH in the neutral environment of the cytosol prevents passively exiting of the cell. Membrane bound carrier proteins at the basel end facilitate removal of IAA from the cell. IAA will again pick up a hydrogen ion, becoming IAAH, and passively move into the next cell below. This chemiotic diffusion is known as basipetal auxin transport or polar auxin transport (Fig. 3). A proton-pump (Fig. 4) is required to maintain pH gradient between intracellular and extracellular environment. The movement of hydrogen ions from the protoplast through the cell wall leads to increased proton concentration, lowering of pH and activation of expansin proteins. Expansin enzymes break the weak hydrogen bonds between cross-linking glycans and cellulose, producing a 'stress relaxation' effect, allowing elongation of microfibrils. 

Figure 3. Polar auxin transport



Figure 4. Proton pump

Historical stages in the discovery of auxin.

• In 1880 Charles Darwin observed the gravitaion of canary grass seedlings towards the only source of available light in his study, which was a small window. Together, with his son, they carried out tests to uncover mechanisms involved and subsequently released a book on phototropism, "The Power of Movement in Plants",  in 1881. See http://www.freeinfosociety.com/media/pdf/4790.pdf for a complete copy.
• Auxin was discovered in 1885 by the German biochemist Ernst Salkowski.
• In 1913, Peter Boysen-Jensen began investigating signals that cause phototropic responses. His simple experiments, which shows the involvement of chemical messengers, involved removing the seedling tip, placing either mica or geletin on the stump, then replacing the tip. 
• Arpad Paal, in 1918, showed build up of chemicals on the oppisite side of the plant, which faces away from the light. He concluded that the tips move this chemical unidirectionally down the shoot.
• In 1926, the Dutch botanist Firts Went showed diffusion of chemicals through agar blocks that had been in contact with the removed tip of a coleoptile. He proved that phototropic response was caused by chemicals moving from the coleoptile tip down the shoot. He called this chemical auxin. See http://bcs.whfreeman.com/thelifewire/content/chp38/3802002.html for Wents experiment.
• Auxin was isolated from human urine by Kogl and Haagen-Smit in 1933.