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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-enhanced cell enlargement
...
7 to 4
...

● It was also understood that it is an energy dependent process and is inhibited by
metabolic inhibitors and inhibitors of auxin-induced growth
...

● Agents other than auxin that can cause proton secretion can also result in growth
promotion
...

36

R
...
Rayle on Auxin
Promoted Growth

This presentation is referenced from:
Plant physiology by Hopkins
...
Cleland and D
...
Cleland and D
...

● This lower pH will activate the cell wall loosening enzymes and help in cell wall
expansion
...
Hager, furthered this theory by adding that auxin caused release of
protons by activating plasma membrane bound ATPase proton pumps
...

● This combined Cleland-Hager proposal is known as acid-growth hypothesis
...
Therefore, there
must be an auxin receptor that initiates a signal transduction pathway
...

38

R
...
Rayle on Auxin Promoted
Growth

Decrease in pH

Turgor mediated cell
expansion
Acidifcation of cell wall

Loosening of cell wall
by cell wall loosening
enzymes

Auxin-activated ATPase-proton pump located in the plasma membrane acidifies the cell wall
space by pumping protons from the cytoplasm
...
Cleland and D
...

● It is designated as ABP1 (Auxin Binding Protein 1)
...

● It is localized mainly in ER but small populations are found in plasma membrane
and in cell wall
...
It also blocks phospholipase A2 activation
...

● Many researchers are unable to find ABP1 in plasma membrane
...

● It contains amino acid sequences at its termini that are present in proteins that are
typically present in plasma membrane
...
There may be an involvement of
possible docking protein
...
Cleland and D
...

● The current proposal stands that there is a lipid soluble anchor for ABP1
...

● It is also proposed that the ABP1-docking protein complex is itself inactive but is
activated by attachment of auxin and starts a signal relaying cascade
...

● Evidence that phospholipase A2 may be involved in this pathway
...
As we studied before, IgGantiABP antibody also blocks phospholipase A 2 activation
...
This effects are inhibited by vandate – a plasma
membrane proton-ATPase inhibitor
...
Cleland and D
...

● Finally, it is also observed that both the IAA and lysophospholipids effects on proton
secretion can be blocked by protein kinase inhibitor
...


Current scheme on how the plasma membrane proton-ATPase is activated by ABP1
and PI3K pathway (involving lysophospholipids)
...
The growth associated with effectiveness after first 30 to 60
minutes
...
It then steadily decreases over next 16
hours
...

The continuous line shows the growth rate when auxin is removed really
early (t = 5 minutes)
...
The growth rate sustains itself for 30
– 60 minutes
...
This shows extensive sustained growth rate even
after 60 minutes and growth being there for around 100 minutes
...
The two curves differ in the
duration of auxin action
...

You can observe the the growth drops off around 60
minutes when auxin is removed very early
...

However, in presence of auxin for extended time,
there is still growth after 60 minutes (where auxin
mediated action of acid-growth stops) and it slowly
tapers for 16 hours
...
The two curves differ in the
duration of auxin action
...

You can observe the the growth drops off around 60
minutes when auxin is removed very early
...

However, in presence of auxin for extended time,
there is still growth after 60 minutes (where auxin
mediated action of acid-growth stops) and it slowly
tapers for 16 hours
...
These includes
SAUR (Small Auxin Upregulated RNAs) and AUX/IAA
...
These RNA transcripts are
detected within 2-3 minutes of auxin application
...


●

AUX/IAA genes are induced over 4 – 30 minutes
...
They function as transcriptional regulators
...
These AUX/IAA
proteins repress the activity of ARFs
...

46

Maintenance of Auxin Induced Growth and Other
Auxin Effects Require Gene Activation
● Additional factors responsible for auxin mediated growth:
● Early studies indicated that many of these responsive genes could also be induced by
the protein synthesis inhibitor cycloheximide (It means that repression of protein
synthesis induced auxin responsive genes)
...
How did this observation came to be?
●
It can be analyzed in a manner that if the genes were controlled at translation level,
they would also be inhibited by cycloheximide
...

●

One model described that auxin initiated ubiquitin mediated degradation of
repressor proteins
...


47

Maintenance of Auxin Induced Growth and Other
Auxin Effects Require Gene Activation
● Additional factors responsible for auxin mediated growth:
● TIR1 is a soluble nuclear located auxin receptor protein
...

● The TIR1 has a recognition site for SCF scaffold and a recognition site for auxin protein
...
However, the affinity of AUX/IAA peptide is very less until and unless auxin
molecule is also present
...

● This TIR1-Auxin-AUX/IAA complex then binds to SCF scaffold and then AUX/IAA is
polyubiquinated and then subsequently degraded by a proteasome
...

48

F – Box (Chapter 17 content)

Role of F-box

● F- box is a component of Ubiquitin-protein ligase
...
Later RBX1, the fourth subunit was
discovered
...

● The F-box have an highly conserved recognition site for
scaffolding protein and a recognition site for target protein
...

● This polyubiquinated protein is then degraded by the
proteasome
...


(1) Auxin response factor protein (ARF) binds to the DNA in the promoter
region of an auxin-responsive gene, but gene transcription is prevented by
the presence of AUX/IAA repressor protein
...
Auxin increases the affinity
ofTRI1 for AUX/IAA and facilitates the dissociation of AUX/IAA from the
ARF (3)
...
Meanwhile, TRI1 recruits
AUX/IAA to the E3 ubiquitin-ligating enzyme, or SCF complex (6), where
(7) AUX/IAA is polyubiquitinated
...
The result is that
when auxin levels are high, TIR1 facilitates active transcription of mRNA
by con

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