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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
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● 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
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● 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 reference
Acid (IAA)

Active acidic
Side chain

Naturally occurring auxins

Synthetic auxins
15

IAA is Synthesized from the Amino Acid LTryptophan
● In 1930s, K
...
Thimann observed the synthesis of IAA in the mold of Rhizopus suinus,
which has been fed tryptophan
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●

What are the drawbacks of such a experiment?
Radio-labeled tryptophan can undergo radio-chemical decomposition and give rise
to IAA by non-enzymatic reactions
...
e
...

The pool size of tryptophan is larger than the synthesized IAA so there is very little
data on actual quantity of IAA synthesized
...

16

IAA is Synthesized from the Amino Acid LTryptophan
● IAA synthesis occurs in three steps
...
The product is Indole-3Pyruvic acid (IPA)
...
This
enzyme removes amino group from variety of tryptophan structural analogs like
phenylalanine and tyrosine
...
This
is carried by the enzyme Indole-3-Pyruvate decarboxylase
...
This reaction is carried
out by NAD dependent Indole-3-Acetaldehyde oxidase
...

4) Finally, IAA can be reversibly converted to Indole-3-Butyric acid (IBA)
...
Seeds of orange pericarp mutant of Zea mays lack enzyme
tryptophan synthase which catalyses final step in tryptophan synthesis
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However, the seedlings have 50-fold higher IAA content than wild
type
...
These mutant plants do no accumulate IAA (like orp mutants) but store IAA
in conjugated form
...

● In both of these mutants it is evident that IAA is synthesized from some precursor other
than tryptophan
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●

The mutants of Arabidopsis accumulate Indole-3-Acetonitrile and also contain nitrilase
enzymes that are necessary to convert Indole-3-Acetonitrile to IAA

● Synthesis of Indole-3-Acetonitrile
...


IAA
Sugar moiety

Glucobrassicin –
A glucosinolate
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● The later was known as bound auxin and has formed chemical conjugates with sugars to
form glycosyl esters
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● Large pool of glycosyl esters are present in seeds of Zea mays
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It is estimated that 60%
of IAA requirement for corn seed is from it’s IAA conjugates
...
It was later discovered that the enzyme peroxidase with
flavoprotein was shown to catalyze the oxidation of IAA
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● The end products of IAA oxidation are physiologically inactive
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aspartate

Irreversible conjugates with
amino acids that are formed in
green tomato fruits
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23

24

Auxin is Involved in Virtually Every Stage of Plant
Development
● Auxin stimulates cell elongation in excised stem and coleoptile sections
...

● Principal test for auxins
...
The
researchers develop a auxin-concentration response curve which shows concentration
dependent relationship to auxin until an optimum concentration is reached
...

● It is also seen that intact stems or coleoptiles do no respond to exogenous application
of auxin
...


25

Auxin is Involved in Virtually Every Stage of Plant
Development
Reduction of growth in
higher than optimal
growth concentrations

Optimal concentration
Peak growth/response

Excised stem used for experimentation

Auxin concentration-response curve
26

Auxin is Involved in Virtually Every Stage of Plant
Development
● Auxin regulates vascular differentiation
...
The stems are
square shaped and have vascular bundle at each corner
...
Normally, the parenchyma
near these vascular bundles differentiate to form new
vascular elements
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This differentiation is controlled by auxin supply
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Removing leaves below wound has no effect
...
It can be observed
when exogenous auxin is used as a supply
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A longitudinal view of regenerated xylem vessel elements around a
wound (W) in a decapitated internode of cucumber (Cucumis sativus)
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1 percent IAA was applied to the upper side of
the internode immediately after wounding
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Coleus stem

Differentiation of phloem sieve tubes is favored by low auxin
concentrations (0
...
0% IAA w/w in lanolin)
...

●

Another experiment showing the role of auxin in vascular differentiation is to cut the
buds and implant the clumps into undifferentiated callus cultures
...


29

Auxin is Involved in Virtually Every Stage of Plant
Development
● Auxin controls growth of auxiliary buds
...
It later separates from the primodia and becomes axillary bud
...

● This dormancy is because the apical bud is able to exert dominant influence and
suppress the cell division and enlargement of axillary bud
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● This was further studied in detail by K
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Thimann and F
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An axillary bud
30

Apical Dominance Studies by
K
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Thimann and F
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Apical Dominance Studies by K
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Thimann and F
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The axillary bud development remained suppressed
in presence of auxin
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● How does auxin from apical tip suppress axillary growth?
The most widely accepted theory is that the optimum concentration of auxin for
axillary bud growth is lower than it is for stem elongation
...

● Further studies using auxin inhibitors like TIBA and NPA allowed more direct evidence
...

● We can also utilize radioactively labeled IAA to observe auxin translocation in real time
...

● Direct measurements of P (using micropressure probe) gave indicated that auxin stimulated
increase in growth rate does not significantly change turgor pressure
...

● However, it is seen that the induction of rapid cell enlargement by auxin is accompanied
by a large change and rapid increase in wall extensibility
...
This is applicable to not living tissues but also to heat killed tissues and
isolated cell walls
...

● The experiments gave rise to the concept of cell wall loosening enzyme with low pH
optimum
...

● How do expansins work?
● The first possibility is that the expansins hydrolyze the cross-linking glycans or other
elements of cell wall matrix that holds cellulose microfibrils in place
...

● The second possibility is that the expansins weaken the covalent bond by which crosslinking glycans are attached to cellulose microfibrils
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The cellulose microfibrils
would then be displaced in response to turgor until tension is reestablished in the crosslink and new cross-links are formed
...

Apical segments (5mm) from darkgrown oat coleoptiles (Avena sativa)
were floated on buffers at the
indicated pH
...

35

The Acid-Growth Hypothesis Explains Auxin
Control of Cell Enlargement
● How is Acid-Growth concept related to auxin?
● During the discovery of acid growth concept, it was also observed that auxin would
cause growing cells to excrete protons
...

●
For example, with the application of auxin the cell wall of Avena coleoptiles
dropped from 5
...
7 in 8 to 10 minutes
...

● Another experiment discovered that if the cell wall space was flooded with neutral
buffers to prevent pH change, the auxin induced growth was prevented
...
For example, Fusicoccin – a phytotoxin from fungus Fusicoccum amugdali
...
Cleland and D
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R
...
Rayle on Auxin Promoted
Growth
● What was the theory provided by R
...
Rayle for auxin stimulated growth?
● They explained that auxin caused acidification of cell walls by stimulating excretion of
protons and this stimulated the increase in cell wall extensibility
...

● At the same time A
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The resultant
increase in cell-wall extensibility allows for turgor induced cell expansion
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● Auxin does not bind to ATPase proton pumps but does enhance it’s activity
...
A putative auxin
receptor has been isolated from Zea mays but the details of the whole pathway are still
obscure
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Cleland and D
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The lower pH activates wall-loosening
enzymes, such as extensins, that loosen the load-bearing bonds
39

R
...
Rayle on Auxin Promoted
Growth
● Auxin receptor of Zea mays
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● It is a glycoprotein dimer of 43 kDa with 22kDa subunits
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● It is seen that IgG-antiABP inhibit auxin induced coleoptile elongation and hyper
polarization of plasma membrane
...

● Some controversies regarding ABP1 as a prime candidate for auxin receptor
...
However, more sensitive
immunolocalization techniques have found small populations in the plasma membrane
...
Moreover, based on amino acid sequence, ABP1
has no lipophilic membrane spanning domain
...

40

R
...
Rayle on Auxin Promoted
Growth
● Some controversies regarding ABP1 as a prime candidate for auxin receptor
...
This ABP1docking protein complex is exported from ER to the plasma membrane where it is
inserted with ABP1 facing outside
...
Some suggests that
the docking protein may belong to the family of G proteins
...

● Other participants that may be involved in this cascade, for example, auxin also activates
phospholipase A2 and it may be involved in this cascade
...

● It is also observed that products of PLA2 – lysophospholipids a
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
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Sheldrake and J
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Raven
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●
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
...
Based on lipid solubility, some amount of protonated IAA (IAAH) will slowly
diffuse across the the plasma membrane into the cell
...

● Inside the cell, the pH is 7
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● The key aspect here is the carrier located at the basal membrane of the cell
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●
This was found by isolating NPA binding protein
...


58

Many Aspects of Plant Development are Linked to
the Polar Transport of Auxin
The chemiosmotic-polar diffusion model for polar transport of
IAA
...
5) approximately 20% of the
IAA is protonated
...
Inside the cell (pH 7
...
IAA− can exit the cell only through efflux carriers
of the PIN family (squares) which are located preferentially at the
base of the cell
...
The uniquely basal location
of the efflux carriers is the key to polar transport
...

●

Two studies on Arabidopsis mutants were carried out and the influx and efflux carriers
were identified
...
The mutations in AUX1 gene locus
causes auxin resistant root growth, reduced lateral root initiation and reduced response to
root gravity
...
The sequence
matches with the known amino acid permeases
...
This (and the similarity of IAA to the amino acid
tryptophan) suggests that the AUX1 is a auxin/proton symport carrier
...

●

NAA uptake by cells is not carrier-mediated, so the loss of AUX1 does not interfere
with the response
...

●

A family of genes called the PIN genes encode putative auxin efflux carrier
...
The first to be discovered was PIN1 which
controls flower development
...
Polar auxin transport is severely reduced in PIN1
mutants
...


●

AUX1 and PIN1 proteins are located at opposite ends of young root phloem cells
...
PIN2 is also localized
in basal membrane
...


62

Many Aspects of Plant Development are Linked to
the Polar Transport of Auxin
● Other roles of PIN proteins
...

●

●

Immediately after the first division of zygote, PIN proteins are located acropetally
in the basal cell, directing the flow of auxin to the apical cell
...

As apical cells further divide, they start to form auxin themselves
...
This develops the
root pole
...




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