the presentation encompasses auxin synthesis, conjugation, degradation, polar and lateral transport and signalling and how all of these together have a bearing on programming and design of the whole plant

1. SUDERSHAN MISHRA (51063)
M.Sc. (Ag) Previous
Deptt. Of Plant Physiology
College of Basic Sciences and Humanities
GBPUA&T, Pantnagar, US Nagar.
A MORPHOGEN IN
PLANT GROWTH AND DEVELOPMENT

2. 2

3.  Discovery of auxin
 Actions of auxin
 Approaches for dissecting mechanisms of auxin action
 3 pillars of morphogenic functionality of auxins
 PIN trafficking
 Foremost action of auxin- cell elongation
 Lateral redistribution of auxins and tropic movements
 Morphogenic effects of auxin
 Case study
 Conclusion
3

4. Fig-1a Early experiments in auxin research
A. From experiments
on coleoptile photo-
tropism, Darwin
concluded in 1880 that
a growth stimulus is
produced in the
coleoptile tip and is
transmitted to the
growth zone.
B. In 1913, P. Boysen-
Jensen discovered that
the growth stimulus
passes through gelatin
but not through water-
impermeable barriers
such as mica.
4

5. Fig-1b Early experiments in auxin research
A. In 1926, F. W. Went
showed that the active
growth promoting
substance can diffuse
into a gelatin block.
B. He also devised a
coleoptile- bending
assay for quantitative
auxin analysis.
5

6. Auxin- The Growth Hormone
Fig-2 Auxin stimulates the elongation of oat coleoptile sections. These
coleoptile sections were incubated for 18 hours in either water (A) or auxin (B)
6

7. Actions of Auxins
CELL
ELONGATION
Growth of Stem
Growth of coleoptiles
Growth of roots
PLANT TROPISMS
Phototropism
Gravitropism
Thigmotropism
DEVELOPMENTAL
EFFECTS
Apical Dominance
Floral Bud Development&
Phyllotaxy
Arrangement of RAM and
SAM
Adventitious root
development
Vascular Differentiation
Role in Abscission
Fruit Development
7

8. Method Sensitivity Specificity Resolution Comments
Mass Spectroscopy-
Molecules identified based
on mass and charge
Medium High Tissue or
organ
L:evel
Can discriminate
between different forms
of Auxin
Immunodetection-
Antibodies recognize
molecules having a specific
confirmation
High Medium Cellular Sensitivity depends on
the accessibility of auxin
to antibody and
specificity of antibody
Reporters- Auxin activated
promoter fused to gene that
produces a visible readout
High High Cellular A transcription-based
synthetic reporter, DR5,
consisting of multiple
tandem repeats of ARF
binding site (TGTCTC)
fused with GFP is used
commonly
Screens- Identification and
characterization of mutants
resistant to exogenous auxin
and auxin polar transport
inhibitors.
High High Cellular
and sub-
cellular
auxin-resistant mutants
at eight loci (aux1; axr1,
2, 4, 5, and 6; Dwf; and
tir1) have been isolated
Approaches used for dissecting mechanisms
of auxin action
Table-1 Various approaches to dissect mechanisms of
auxin action
8

9. Approaches used ………….(Contd.)
Developing Reporters Mutant Screens
 Analysis of the promoter regions of
auxin inducible genes
 Identify the transcription factors
binding to the AuxREs
 Identify the conserved elements: e.g.
TGTCTC
 Fuse multiple copies of the conserved
elements to visible readout signal like
GFP or GUS
 Auxin-resistant mutants at eight loci
(aux1; axr1, 2, 4, 5, and 6; Dwf; and
tir1) have been isolated by their ability
to elongate roots on normally
prohibitive concentrations of auxin
9

10. AUXIN HOMEOSTASIS
BIOSYNTHESIS , CONJUGATION AND DEGRADATION
AUXIN TRANSPORT
POLAR, AND REDISTRIBUTIONARY , PIN TRAFFICKING
AUXIN SIGNALLING
3 Pillars of Morphogenic Functionality of
Auxin
10

11. Auxin Homeostasis- biosynthesis
Fig-3a Trp Dependent auxin biosynthesis pathways
11

12. Auxin Homeostasis- biosynthesis
Fig-3b Trp independent auxin biosynthesis pathways
12

13. Auxin Homeostasis- conjugation
Fig-3c Different conjugation products of auxin
13

14. Auxin Homeostasis- degradation
Fig-3d Various degradation pathways of auxin
14

15. Auxin Transport- Polar transport of Auxin is
gravity independent
Fig-4a Simple experiment to demonstrate that polarity of
auxin transport is independent of gravity
Fig-4b Roots grow from the
basal ends of these bamboo
sections, even when they are
inverted. The roots form at
the basal end because polar
auxin transport in the shoot
is independent of gravity
15

16. Auxin Transport- Chemiosmotic mechanism
of polar auxin transport
AUXIN INFLUX
 Passive diffusion of the protonated
(IAAH) form across the phospholipid
bilayer
 Secondary active transport of the
dissociated (IAA–) form via a 2H+–IAA–
symporter
 These symporters are a family of
AUX/LAX permeases related to bacterial
amino acid carriers
 This is saturable and specific for active
auxins
AUXIN EFFLUX
 Most of the Auxin
escapes via auxin
anion efflux carrier
 A family of putative
auxin efflux carriers
known as PIN
proteins (named
after the pin-shaped
inflorescences
formed by the pin1
mutant of
Arabidopsis are
localized precisely at
the basal ends of the
conducting cells
Fig-4c pin1
mutant of
Arabidopsis
16

17. Chemiosmotic mechanism……..(Contd)
Fig- 4d The repetition of auxin uptake
at the apical end of the cell and
preferential release from the base of
each cell in the pathway gives rise to the
total polar transport effect.
17

18. Integrated model of polar auxin transport
Fig-4e Schematic representation
of Polar auxin transport
 In addition to the well known
AUX1/LAX and PIN family proteins
it includes a family of ATP
dependent transporters
 These are ‘P’ glycoproteins or ‘B’
subclass of the large ABC family of
integral membrane transporters
 Hence a subgroup of PGP/ABCB
transporters function as ATP
dependent amphipathic auxin
anion efflux carriers
 ABCBs are uniformly distributed
rather than polarly however PINs
function synergistically with ABCB
in polar auxin transport
 Certain tonoplast carriers like
ZIFL1.1 help in stabilizing PIN
proteins particularly pin 2
18

19. Phloem mediated non-polar auxin transport
Fig-4f Asymmetric localization
of AUX1 in a file of protophloem
cells
 The asymmetrically oriented AUX1
permease promotes the acropetal
movement of auxin from the
phloem to the root apex
 The phloem appears to represent
the principal pathway for long-
distance auxin translocation to the
root.
 Long-distance auxin transport in
the phloem is important for
controlling such processes as
cambial cell divisions, callose
accumulation or removal from
sieve tube elements, and branch
root formation.
 Polar and non polar transport are
not independent of each other and
can be exchanged at shoot apices
19

20.  Subcellular polarity of PINs determine the direction of auxin efflux out of that
cell and thus coordinated PIN localizations along the chain of cells channelize
directionality of auxin transport.
 PINs are not statically localized at the plasma membrane but undergo rapid
clathrin mediated endocytic cycling involving PIN internalization from plasma
membrane
 They are recycled back to plasma membrane via ARF-GEF (GNOM) regulated
polar cycling
 The protein kinase PINOID and protein phosphatase 2A complex RCN1 are
also involved in regulating this PIN trafficking
 Auxin efflux inhibitors like TIBA, NPA and certain flavonols affect this
trafficking
PIN trafficking and localization with respect
to directionality of auxin transport
20

21. A B C
D E
Fig-4g (A) Control, showing
asymmetric localization of PIN1.
(B) After treatment with brefeldin
A(BFA). (C) Following an
additional two-hour washout of
BFA. (D) Following a BFA
washout with cytochalasin D.
(E) Following a BFAwashout with
the auxin transport inhibitor
TIBA.
PIN trafficking and localization…..(Contd.)
21

22. PIN trafficking and localization…..(Contd.)
Fig-4i Actin dependent pin
cycling between plasma
membrane and an endosomal
compartment
Fig-4h mechanism of
establishment of PIN polarity by
AGC-3 Kinases
22

23. Auxin Signalling
 Proteins of TIR1/AFB family are the principle auxin receptors
 They are F-box protein components of an SCF-ubiquitin E3 ligase complex
(SCFTIR1/AFB)that catalyzes the ATP dependent covalent addition of Ubiquitin
molecules to proteins targeted for proteolytic degradation
 Auxin acts as a molecular glue to bring together a heterodimer consisting of
one TIR1/AFB and one AUX/IAA protein
 AUX/IAA proteins are one of the two families of transcriptional regulators,
the other being the ARFs. Both are antagonists to each other. If the ARF in
question is a transcriptional activator, the corresponding AUX/IAA protein
will act as repressor
 ARFs are short lived nuclear proteins that bind DNA AuxREs containing the
consensus sequence TGTCTC in promoters of auxin response genes
 AUX/IAA bind to ARF proteins bound to DNA
23

24. Auxin Signalling
Fig-5 Auxin regulation of transcriptional activation of early response
genes
24

25. Foremost effect of auxin- cell elongation
 Auxin Rapidly Increases the Extensibility
of the Cell Wall
 The effects of various parameters on the
growth rate are encapsulated in the
growth rate equation:
GR = m (ψp – Y)
 Where GR is the growth rate, Yp is the
turgor pressure, Y is the yield threshold,
and m is the coefficient (wall
extensibility) that relates the growth
rate to the difference between ψp and Y.
 Auxin-Induced proton extrusion
acidifies the cell wall and increases cell
extension. This proton extrusion my
involve both activation as well as
synthesis of plasma membrane H+ -
ATPases.
 ABPs and Expansin proteins are involved
Fig-6Kinetics of auxin-induced elongation
and cell wall acidification in maize
coleoptiles. The pH of the cell wall was
measured with a pH microelectrode. Note
the similar lag times (10 to 15 minutes) for
both cell wall acidification
and the increase in the rate of elongation
25

26. Lateral redistribution of auxins- the primary
cause of plant tropisms
Fig-7 Evidence that the lateral redistribution of auxin is stimulated by unidirectional
light in corn coleoptiles
26

27. Lateral redistribution …….plant tropisms
(Contd.)
Fig-8 Visualization of lateral redistribution using A and B Agar blocks C.. DR5::GUS
reporter gene construct
(c)
27

28. Fig-9 Statoliths and Redistribution of auxin during gravitropism in
maize roots
Lateral redistribution …….plant tropisms
(Contd.)
28

29.  Commencement of polarized auxin transport in globular embryo might
facilitate the morphological polarity expressed in subsequent stages of plant
embryogenesis
 Once the first division of zygote is over, PIN7 is confined to the apical side of
the basal cell driving auxin transport into apical cell
 At 32 cell phase PIN7 polarity reverses to the basal membrane of the
suspensor cells
 PIN4 regulates transport in hypophysis while PIN 3 in columella precursors
 Single pin mutant dosen’t display darmatic effects but miltiple pin mutants
show severe root and shoot pole defects
Auxin as a morphogen during
embryogenesis
29

30. Auxin……….embryogenesis (contd.)
Fig-11 Polar-localized auxin efflux carrier PIN proteins direct auxin flow
during embryogenesis and root meristem growth to generate local auxin
accumulation foci responsible for organ growth
Fig-10 Immunolocalization
of AUX1 in protophloem
cells of the stele, a central
cluster of cells in the
columella, and
lateral root cap cells
30

31. Auxin……….embryogenesis (contd.)
Fig-12 Genes whose functions are essential for Arabidopsis
embryogenesis
A. GNOM (GN)
Encodes a guanine
nucleotide exchange
factor (GEF) which
enables polar
distribution of auxin
by establishing a
polar distribution of
PIN efflux carriers
B. MONOPTEROS (MP)-
necessary for the
normal formation of
basal elements such
as the root and
hypocotyl , encodes
and auxin response
factor
31

32. Auxin and shoot morphogenesis
 Auxin is sufficient in itself to trigger organ initiation when applied at the tip
of naked meristem of pin1mutant which is defective in polar auxin transport
 Modular studies incorporating polar transport of the auxins and mechanical
strains suggest that auxin is transported to the site of lateral organ
primordia inception by polar auxin efflux carrier PIN1
 Growing lateral organ primordium acts as sink and this leads to depletion of
auxin from surrounding cells, creating an inhibitory field which in turn
controls the spacing between lateral organs to define a specific phyllotactic
pattern.
 Recently it has been shown that pin1 hypomorphs result in switch of spiral
pattern to opposite pattern (Prasad et al. 2011)
32

33. Auxin and shoot morphogenesis (Contd.)
Fig-13 Polar auxin transport based schematic representation of different patterns
arising at the shoot apex; (a) Distichous (alternate), (b) Spiral, (c) Decussate
(opposite) pattern. I (Incipient primordium), P (Primordium)
33

34.  When the apical bud is grafted onto a
clump of undifferentiated cells, or
callus, xylem and phloem
differentiate beneath the graft.
 The relative amounts of xylem and
phloem formed are regulated by the
auxin concentration: High auxin
concentrations induce the
differentiation of xylem and phloem,
but only phloem differentiates at low
auxin concentrations
 The regeneration of vascular tissue
following wounding is also controlled
by auxin produced by the young leaf
directly above the wound site
 Vascular differentiation is polar and
occurs from leaves to roots.
Auxin and Vascular Differentiation
Fig-14 Detection of sites of auxin
synthesis and transport in a young leaf
primordium of DR5 Arabidopsis by
means of a GUS reporter gene with an
auxin-sensitive promoter.
34

35. Auxin and Vascular Differentiation (Contd.)
Fig-15 IAA-induced xylem regeneration around the wound in cucumber (Cucumis
sativus) stem tissue. (A) Method for carrying out the wound regeneration experiment.
(B) Fluorescence micrograph showing regenerating vascular tissue
around the wound35

36. Auxin and Root Development
 Although elongation of the
primary root is inhibited by
auxin concentrations greater
than 10–8 M, initiation of
lateral (branch) roots and
adventitious roots is
stimulated by high auxin levels
 A series of Arabidopsis
mutants, named alf (aberrant
ateral root formation), have
provided some insights into
the role of auxin in the
initiation of lateral roots. The
alf1 mutant exhibits extreme
proliferation of adventitious
and lateral roots, coupled with
a 17-fold increase in
endogenous auxin
Fig-16 Root morphology of Arabidopsis (A–C) wild-type
and alf1 seedlings (D–F) on hormone-free medium. Note
the proliferation of root primordia growing from the
pericycle in the alf1 seedlings (D and E)36

37.  Patterning an angiosperm flower requires
the combined and individual functions of
class A, B, C, D, and E MADS box
transcription factors
 LOFSEP group of genes are specific to
grasses and contribute to panicle
morphology, spikelet and FM specification,
floral organ differentiation, and meristem
determinacy
 Rice LEAFY HULL STERILE (LHS1)/OsMADS1,
a member of the grass subgroup of LOFSEP
genes, referred to henceforth as OsMADS1,
is expressed in FMs, functions during FM
establishment and floret organ patterning,
and contributes to meristem termination
Auxin as a morphogen in floral meristem
specification
Fig-17 OsMADS1 exerts its effects through
meristem regulators and activating auxin
signaling
37

38.  Title- “Auxin Acts through MONOPTEROS to
Regulate Plant Cell Polarity and Pattern
Phyllotaxis”
 Neha Bhatia, Behruz Bozorg, Andre´ Larsson, Carolyn Ohno, Henrik Jonsson,
Marcus G. Heisler
Case Study
38

39. Central idea of the study
Organ positioning in plants
depends on polar transport
of the hormone auxin to
organ initiation sites. The
auxin response factor
MONOPTEROS orients the
polarity of the auxin efflux
carrier PIN1 non-cell
autonomously, thereby
facilitating a positive
feedback loop that results in
periodic organ formation.
39

40.  Auxin-regulated MP expression and activity predict
PIN1 polarity changes at the SAM
 Localized MP activity is necessary to mediate periodic
organ formation
 MP orients PIN1 polarity non-cell autonomously to
promote local auxin accumulation
 Sub-epidermal MP activity is required to stabilize auxin
distribution patterns
Highlights of the study
40

41. Results
41

42. Figure 18. MP Expression
Patterns Predict PIN1 Polarity
Changes
(A) pMP::MP-YPet (green) and
pPIN1::PIN1-CFP (magenta)
expression and localization in
the mp-T370 inflorescence
meristem (IM).
(B) Meristem in (A) showing
pPIN1::PIN1-CFP alone.
(C) Meristem in (A) showing
pMP::MP-YPet expression alone.
(D) Magnified view of i4 before
PIN1 polarity convergence.
(E) Magnified view of i3 after
PIN1 convergence.
(F) Magnified view of i1. Note
the low MP expression
surrounding i1 prior to PIN1
polarity reversal.
(G) Magnified view of P1
showing reduced MP expression
surrounding the primordium
and PIN1 polarity oriented away
from low-MP-expressing cells.
The arrows indicate the
estimated PIN1 polarity
direction within the cells.
Primordium (P) and incipient
primordium (i) stages are
numbered i4–P5
42

43. Figure 19. Localized Organogenesis
Requires
Localized MP Activity
(A) Wild-type seedling 18 days after
induction
of pUBQ10>>MPc794-YPet (dotted
rectangle) in
comparison to an un-induced plant.
(B) Magnified view of an induced
plant from (A).
Note the fusion of the first two
leaves.
(C–H) pPIN1::PIN1-CFP expression
and polarity
(green) after induction of
pUBQ10>>MPc794-Ypet
(I–K) pPIN1::PIN1-CFP expression and
polarity
(green) before (I) and after induction
of
pUBQ10>>MPc794-YPet (magenta) (J
and K) in
mp IM. Note the ring-shaped organ
present 5 days
after MPc794-YPet induction (K).
The asterisk in (C) marks the
removed cotyledon.
43

44. Figure 20. MP Polarizes Cells Non-Cell
Autonomously
(A and B) Confocal projection showing
the mp-B4149 apex expressing
pPIN1::PIN1-GFP (magenta) before (A)
and 8 days after induction of MP-YPet
clones (B) C) Frequency of peripheral
MP clones associated with PIN1
convergence patterns (n = 60
peripheral clones of MP). (D–M) Time
series showing epidermal MP-VENUS
clones in mp-T370 and associated
changes in PIN1-CFP localization.
(D–H) Overview showing two
independent clones. . (I–M) Magnified
view of the dotted rectangle in (D).
(N) Longitudinal optical section
showing a sub-epidermal polarity
response to the epidermal clone.
(O) Cross-section showing lateral PIN1
polarity toward the MP clone in the
sub-epidermal layer.
(P) PIN1-CFP convergence in the
epidermal cell layer in response to a
sub-epidermal MP clone.
(Q) Longitudinal reconstructed section
of (P) showing a sub-epidermal MP-
VENUS clone.
The arrows indicate the estimated PIN
polarity direction within the cells.
44

45. Figure 21. Restriction of MP Activity
to the Epidermis Results in Mobile
Auxin Maxima
(A and B) pML1::MP-YPet expression
(magenta) in wild-type before (A)
and 6 hr after auxin treatment (B) (n
> 20).
(C and D) pML1::2X-CFP-N7
expression (magenta) before (C) and
6 hr after auxin treatment (D) (n =
6).
(E) Photograph of the mp-T370
mutant expressing pML1::MP-YPet.
(F) Confocal projection of an mp-
T370 mutant seedling with fused
leaves (white arrowhead), with
pML1::MP-YPet (magenta) and
pPIN1::PIN1-CFP (green).
(G and H) IM of mp-T370
expressing pML1::MP-YPet;
photograph (G) and confocal
projection, with pML1::MP-YPet
(magenta) and pPIN1::PIN1-CFP
(green) (H).
(I) Magnified view of the dotted
rectangle in
(J and K) Time series of mp-T370
IMs expressing pML1::MP-YPet and
pPIN1::PIN1-CFP before (J) and 12 hr
later (K). (L) or the absence thereof
(M).
45

46. Towards the end
46

47. Fig- 22 Cross-regulatory interactions of different phytohormones
with auxin for understanding the intricacies involved in plant root
development.47

48. Fig-23 Cross-regulatory interactions at molecular level
48

49.  Cell division increases only the number of cells and cell
expansion but growth is ultimately reflected in tissue
morphology, organ shape and plant architecture
 Auxin signaling relates to auxin distribution patterns which is
in turn related to landscape of cell to cell polar auxin
transport canals
 Polar transport leads to cell polarity that governs organ
polarity and has immediate bearing on the morphogenetic
patterns
 Hence auxin because of its directionally regulated
concentration and signaling poses as the most potent plant
morphogen
Conclusion
49

50. 50