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Auxin
Aman Bisht
The Maharaja Sayajirao University of Baroda

This presentation is referenced from:
Plant physiology by Hopkins
...

● They are signal molecules that act individually or cooperatively to carry information
between cells
...

● These few slides will try to explain:
●

Hormone biochemistry – What kind of molecules these hormones are
...


●

Hormone metabolism and biosynthesis
...
A huge amount of studies
were done to understand hormones, their action, their relation to disease, etc
...
H
...
It means to excite or
arouse
...
He observed the formation of roots on the swellings that occur above
girdle wounds that interrupted the phloem tissues around the stems of woody plants
...
He
postulated that root forming substance, produced in leaves would migrate down in the stem to
initiate root formation
...
W
...

● It was H
...

2

Observations of Duhamel du Monceau

Girdling on a plant

Root formation in girdled stem

3

The Hormone Concept in Plants
● What are hormones?
● These are naturally occurring organic molecules present at low concentrations that
influence the physiological processes
...

●
Transported in the bloodstream to a specific target tissue
...

● Unlike animal hormones, the site of synthesis and mode of transport of plant
hormones are not always clearly localized
...

● Hormones are short lived and serve a regulatory role
...

4

The Hormone Concept in Plants
● Hormone pool and the availability of hormone
...
The amount of
hormone available will be governed by the rate at which an active hormone molecule
enter and exit the hormone pool
...

●
Retrieval of a active hormone from an inactive storage form
...

● The removal of hormone is through:
●
Oxidation or some other form of chemical degradation
...
(See – Slide 25)

5

Auxin is Distributed Throughout the Plant

Auxin distribution in an oat
seedling (Avena sativa),
showing higher
concentrations of hormone
in the actively growing
coleoptile and root apices
...
It was the
first plant hormone to be
discovered
...

● It is synthesized in meristematic
regions and other actively growing
organs such as coleoptile apices, root
tips, germinating seeds and the
apical buds of growing stems
...

6

Discovery of Auxin

This presentation is referenced from:
Plant physiology by Hopkins
...

● One of the movements he studied was the tendency of canary grass (Phalaris canariensis)
seedlings to bend towards light coming from the window
...

● The primary leaves of grass seedlings are enclosed in a hollow sheath like structure
called coleoptile
...

● Darwin observed that coleoptile, like stem grow towards the light source
...
However this
curvature did not happen if the tip of the coleoptile was removed or covered to
exclude light
...


8

Discovery of Auxin

Darwin experiment
Boysen-Jensen
experiment

Darwin said that “when the seedlings are freely exposed to lateral
light, some influence is transmitted from the upper to the lower part,
causing the latter to bend
...

● In 1918, Paal showed that if the apex were removed and replaced asymmetrically,
curvature would occur even in darkness
...
W
...

● Went removed the apex of oat (Avena sativa) coleoptile and stood the apical pieces in
small blocks of agar
...
The substance
then diffused from the block into the coleoptile, preferentially stimulating elongation of the
cells on the side of the coleoptile below the agar block
...

● The curvature proved to be proportional to the amount of active substance in the agar
...
(See – Slide 14,15)
Because of his incredible work, intensive efforts were put to isolate the compound
...

At the same time, IAA was isolated from yeast extracts, following year, from cultures of Rhizopus
suinus
...

11

Discovery of Auxin

Auxin preferentially stimulates cell growth
when the agar block is placed asymmetrically

Avena Curvature Test
12

Discovery of Auxin

Since there is reduction in
growth after optimal auxin
concentration, we see no
increase in curvature after
the peak optimal auxin
concentration

The graph

Avena Curvature Test

1

The apex of plant
coleoptile
is removed
...


2

The agar block is placed
asymmetrically on another
excised tip
...


13

The Principal Auxin in Plants is Indole-3-Acetic
Acid (IAA)
● Indole-3-Acetic acid is the most widely distributed natural auxin
...
However, most of these compounds occur as a
precursors to IAA
...

● The unifying character of these molecules is that they have an acidic side chain on an
aromatic ring
...
For example,
● In vegetative tissue, the IAA concentration ranges from 1μg and 100 μg (5
...

● The seeds have much higher amount of IAA in comparison (for rapid growth)
...
V
...

● The classical experiment to study the synthesis of IAA is to feed plants radioactive labeled
tryptophan and examining residual radioactivity in isolated IAA
...
This will skew the data and it’s interpretation as
to what amount of IAA is produced by the plant (i
...
via enzymatic reactions)
...

The best these experiments provide is the fact that it clearly establishes that plants
are able to synthesize IAA from tryptophan
...

1) The removal of amino group from tryptophan side chain
...
This reaction is catalyzed by tryptophanamino transferase
...

2) The second step is decarboxylation of IPA into Indole-3-Acetaldehyde (IAAld)
...

3) In the last step IAAld is oxidized to Indole-Acetic acid (IAA)
...

Note that IAAld may also be reversibly reduced to indole-3-ethanol
...

17

IAA is Synthesized from the Amino Acid LTryptophan

Indole-3Acetaldehyde
oxidase

Indole-3-Pyruvate
decarboxylase

Tryptophanamino
transferase

NAD dependent

18

Some Plants Do Not Require Tryptophan for IAA
Biosynthesis
● IAA can be synthesized via tryptophan independent pathway (observed from plant mutants
of maize and arabidopsis)
...
These orp
mutants do germinate but do not survive for long because of their diminished capacity for
tryptophan synthesis
...

● Other tryptophan mutants have been discovered like trp2 and trp3 isolated from Arabidopsis
(These also lack tryptophan synthase and are unable to convert Indole-3-Glycerol phosphate
to tryptophan)
...

● These Arabidopsis mutants accumulate Indole-3-Acetonitrile
...

19

Some Plants Do Not Require Tryptophan for IAA
Biosynthesis
● Reversible conversion of IAA to Indole-3-Acetonitrile
...

●

Indole-3-Acetonitrile can be derived from glucobrassicin, a principle glucosinolate in
the members of family Cruciferae
...

20

IAA may be Stored as Inactive Conjugates
● Early in research of hormones, two populations of auxins were recognized – one was free
moving can could diffused into agar (look into Avena curvature test) and the other could only
be isolated through solvent extraction or via alkaline hydrolysis
...

●

These conjugates are usually formed by esterification of glucose or inositol molecule to
the acid group of the side chain
...
These are formed in the milky
endosperm and are important source of active hormone for the seed
...


21

IAA may be Stored as Inactive Conjugates
Inositol

aspartate

Irreversible conjugates
Reversible conjugates
22

IAA may be Stored as Inactive Conjugates
● The enzyme responsible for inactivating IAA was first isolated from plant extract in 1940s,
and was called IAA oxidase
...
This reaction releases CO2
...

● It is also seen that green tomato fruits (Vicia faba), conjugate IAA with amino acids like
alanine and aspartic acid which leads to irreversible deactivation
...

It is yet another process for IAA
inactivation
...
Other functions of
auxin involves – secondary root formation, vascular differentiation, development of
auxiliary buds, flowers, and fruits
...

● The principal test for auxin is stimulation of cell enlargement in excised tissues
...

Concentration exceeding the optimum result in reduced growth
...
Thus the research usually involves experimentation on excised segments of
stem or coleoptiles
...

●

The favorite system to study vascular differentiation is Coleus stem
...

●

●

Coleus stem cross section

●

A wedge shaped incision is made that interrupts one
of the vascular bundles
...
These will then reestablish the
severed connection
...
If the
leaves are removed above the wound (the leaves are
the source for auxins), the vascular regeneration is
reduced
...

27

Auxin is Involved in Virtually Every Stage of Plant
Development
●

It is also observed that the extent of vascular regeneration is
directly proportional to the auxin supply
...


IAA-induced xylem regeneration
...

Lanolin containing 0
...
Polar regeneration is
indicated by the dense appearance of many xylem tracheary elements
(arrow) in the region of the damaged vascular bundle above the wound
...
1% IAA w/w in lanolin) while xylem differentiation
is favored by higher auxin concentrations (1
...

28

Auxin is Involved in Virtually Every Stage of Plant
Development
● Auxin regulates vascular differentiation
...
It can be seen that
auxin causes vascular differentiation in these calluses
...

● As the shoot continues to grow, the apical meristem lays down new leaf primodia, a group
of cells called the axil
...

This axillary bud usually remains dormant but will resume growth if the shoot apex is
removed
...
This is called apical
dominance
...
V
...
Skoog
...
V
...
Skoog

This presentation is referenced from:
Plant physiology by Hopkins
...
V
...

Skoog
● Is apical dominance controlled by auxin?
● Thimann and Skoog tested this idea by decapitating broad bean (Vicia faba) plants and
applying auxin to the cut stump
...

● In absence of auxin, the axillary bud growth was promoted
...

● The stream of auxin flowing from shoot apex to axillary bud maintains the
inhibitory concentration of auxin in the axillary bud
...

These were applied in between the shoo apex and the axillary bud
...

●

32

The Acid-Growth Hypothesis Explains Auxin
Control of Cell Enlargement
● Any type of cell expansion will follow the following equation:
dV\dt = Change in volume over time, Y = Yield threshold, m = Cell
wall extensibility, P = Turgor pressure
...
Indirect
experiments also establish that the yield threshold does not change significantly either
...


33

Wall Loosening and Cell Expansion is Stimulated
by Low pH (Chapter 17 content)
● It has been known since 1930s, that plant tissues elongate faster when bathed in a medium
with low pH
...
This phenomenon is called acid-growth phenomenon
...

● Expansins are the proteins that induce stress relaxation and cell wall extension at low pH
...
However, this
has not been demonstrated
...
They partial detachment of a
xyloglucan that was under tension would allow it to relax
...


34

Wall Loosening and Cell Expansion is Stimulated
by Low pH

An experiment demonstrating the
acid-growth response
...

The increase in length of the
segments was measured after 18
hours
...
Several evidences point out that that this
phenomena is crucial for auxin-enhan
SAUR genes or AUX/IAA genes) and act to activate or repress gene expression
...

● This observation suggests that these genes may be controlled by short lived repressor that
normally prevents transcription
...
If there are genes that respond to
inhibition of protein synthesis then it means that there is a protein that is
inhibiting transcription which gets inactivated or produced below threshold
value during inhibition of protein synthesis
...
This model was confirmed with the discovery of TIR1 gene in
Arabidopsis
...
It works in conjunction with
auxin to de-repress transcription (activation) of auxin responsive genes
...

● Crystal structure has shown that TIR1 has a pocket that accommodates AUX/IAA
peptide
...

● Auxin serves the role of a molecular glue that enhances AUX/IAA interaction with
TIR1 protein
...

● Auxin appears to modulate development through depression of auxin-responsive
genes, not through a simple activation
...
The most
common ubiquitin protein ligase in plants is SCF complex,
named after the first three protein subunits discovered – Skp1,
Cullin, and F-box protein
...

● F-boxes have unique substrate specificity and attach to Skp1,
Cullin and RBX1 which form the scaffold protein for F boxtarget protein binding
...

● One F-box has has delivered the target protein to the scaffold,
the now complete SCF complex then facilitates the transfer
of the ubiquitin from E2 to the target protein
...

49

Maintenance of Auxin Induced Growth and Other
Auxin Effects Require Gene Activation
A model for auxin-induced gene de-repression
...
When auxin levels are
elevated, the auxin (A) combines with a nuclear-located auxin receptor,
TRI1, to form an auxin-TRI1 complex (2)
...
Removal of the AUX/IAA proteins from the ARF derepresses the
gene (4), allowing transcription of mRNA and the translation of auxininduced proteins, including AUX/IAA (5)
...
The ubiqitinated protein is then
recruited to the 26S proteasome (8), where it is degraded
...
When auxin levels are low,
TIR1 is unable to bind with the repressor, the repressor protein
accumulates, and transcription is shut down
...

● There is a highly polar transport
mechanism involved in this transport
...

● This movement, when from the
opposite direction, is referred as
acropetal
...

Receiver block is on
bottom
...

Transport √

Donor block is on
bottom
...

The orientation is from
apical to basal
...

Receiver block is on top
...

Transport X

Donor block is on top
...

The orientation is from
basal to apical
...
Acropetal transport in shoots is
minimal
...
The acropetal system coming from the shoots
...
There also exists a basipetal
system in roots which reverses the flow of auxin, moving it from root tip to the central
cylinder
...
The flow of auxin in roots in
intimately connected with the root response to gravity
...

● For example, polar transport is inhibited by anaerobiosis an chemicals like cyanide and
2,4-dinitrophenol
...

● Certain chemicals called phytotropins (TIBA (2,3,5-triiodobenzoic acid)), morphactin
(9-hydroxyfluorine-9-carboxylic acid) and NPA (N-1-naphthylphthalamic acid)) are noncompetitive inhibitors of auxin transport
...

● Lastly, Radioactive IAA and Non-radioactive IAA compete with each other during
transport
...

55

Many Aspects of Plant Development are Linked to
the Polar Transport of Auxin
You can clearly observe that TIBA and NPA would not bind
competitively as they do not look remotely similar to IAA
...

●

This theory was provided by P
...
Rubery, A
...
Sheldrake and J
...
Raven
...

●
An influx carrier protein is required for the entry
...


● The process of transport
...
Depending on pH it may exist in a
protonated form (IAAH) or in unprotonated form (IAA-)
...
5
...

57

Many Aspects of Plant Development are Linked to
the Polar Transport of Auxin
● The process of transport
...


63

FIN

This presentation is referenced from:
Plant physiology by Hopkins

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