Part II: Phytohormones and Tropisms

1
Part II Phytohormones and Tropisms

• Form and function of multicellular organisms
  depend on efficient
• communication among cells, tissues and organs
• - Morphogenesis depends on chemical signals
  Julius von Sachs (1832-1897)

Hormone from Greek horman - "to set in motion"
chemical messenger that mediates
intercellular (between cells) communication
interacts with specific cellular proteins
called receptors present in cell in very low
concentrations control plant development and
responses to stress Growth hormones
Stress hormones Auxin Salicylic
acid Gibberellins Jasmonic acid Cytokinins
Oligosaccharines Ethylene Abscisic
acid Brassinosteroids (plant steroid
hormone) Oligosaccharines
2
Lecture 7 Auxin IBiosynthesisMetabolism
Transport
3
The auxin concept A historical perspective
From experiments on coleoptile
phototropism Darwin concluded that a growth
stimulus is produced in coleoptile tip and
transmitted to growth zone.
Growth stimulus passes through gelatin but not
through water-impermeable barriers
Coleoptile sheath/protective organ of the
youngest leaves in grasses
4
The auxin concept A historical perspective
The growth stimulus is of chemical nature.
5
The auxin concept A historical perspective
The growth-promoting substance can diffuse into a
gelatin block.
Coleoptile-bending assay
Auxin IAA
6
Auxin stimulates the elongation of oat
coleoptile sections
primary leaves
coleoptile
Incubation in water for 18h
Incubation in auxin for 18h
auxin from the greek word auxien, meaning to
increase or to grow
7
What is auxin?
Structures of three natural auxins
Indole
All plants
Peas
Mustard and corn
8
Synthetic auxins
Often used as herbicides
9
Auxin biosynthesis

• Synthesis in meristems, young leaves,
• developing fruits and seeds
• GUS (ß-glucuronidase) fused to promoter
• containing an auxin response element
• transform Arabidopsis with construct (Ti
• plasmid) using Agrobacterium
• Visualize production/distribution of auxin
• by GUS staining (expression of GUS
• reporter gene)
• Auxin is produced by a cluster of cells located
  at sites where hydathodes will develop
• Hydathodes grand-like modifications of ground
  and vascular tissues for release of liquid water
  (guttation)

Young leaf primordium
GUS stain location of auxin synthesis
Gradient of diluted GUS activity
Differentiating vascular strand, sink for auxin
Wherever free auxin is produced, GUS expression
(blue staining) occurs
Aloni et al. 2003 Planta 216 841
10
GUS reporter gene
Look for GUS activity
X-Gluc (5-bromo-4-chloro-3- indolyl-beta-D-glucuro
nic acid)
Look for blue staining in tissue
Substrate for GUS
11
Auxin biosynthesis

• Synthesis in meristems, young leaves,
• developing fruits and seeds
• GUS (ß-glucuronidase) fused to promoter
• containing an auxin response element
• transform Arabidopsis with construct (Ti
• plasmid) using Agrobacterium
• Visualize production/distribution of auxin
• by GUS staining (expression of GUS
• reporter gene)
• Auxin is produced by a cluster of cells located
  at sites where hydathodes will develop
• Hydathodes grand-like modifications of ground
  and vascular tissues for release of liquid water
  (guttation)

Young leaf primordium
GUS stain location of auxin synthesis
Gradient of diluted GUS activity
Differentiating vascular strand, sink for auxin
Wherever free auxin is produced, GUS expression
(blue staining) occurs
Aloni et al. 2003 Planta 216 841
12
Tryptophan biosynthetic pathway
Arabidopsis mutants trp2 and trp3 are blocked in
the last two steps of tryptophan biosynthesis
IAN accumulates up to 11-fold in mutants vs. wt
(Radwanski et al. 1996).
In tomato, IPA synthesized independently of
tryptophan (Nonhebel et al. 1993). Depending on
species either IAN or IPA may serve as the
intermediate between either indole-3-glycerol
phosphate or indole, respectively, and IAA
(Arabidopsis can do both).
Web Topic 19.4
13
orange pericarp (orp) mutant of maize
- orp has mutations in both loci encoding
tryptophan synthase - pericarps surrounding each
mutant kernel (orange) accumulate glucosides of
anthranile acid and indole, which is responsible
for the orange color
14
Multiple pathways exist for the biosynthesis of
auxin
Most common
Brassicaceae Poaceae Musaceae
Pathogenic bacteria Pseudomonas
savastonoi Agrobacterium tumefaciens
Tomato has both IPA and TAM pathway
15
Most auxin in plants is in a covalently bound form

• Free IAA is biologically active form
• Majority of auxin found in covalently bound
• form (hormonally inactive)
• Low-molecular-weight conjugated auxins (Fig.)
• High-molecular-weight conjugates IAA-glucan
• (7-50 glucose units per IAA), IAA-glucose-
• proteins found in cereal seeds

16
Auxin is degraded by multiple pathways
Enzymatic (oxidative) breakdown
IAA may be oxidized non- enzymatically in vitro
in high- intensity light (this does not occur in
vivo)
17
Factors regulating the steady-state level of auxin
Web Topic 19.5
18
Auxin concentrations in different regions of the
shoot
IAA in different regions of WT tobacco
Tobacco transformant expressing TRP monoxygenase
and IAM hydrolase from Agrobacterium tumefaciens
to produce IAA throughout the plant.
Auxin concentration in leaves
19
Subcellular localization of auxin

• Distribution of IAA in cell is regulated by pH
• IAA_ does not cross membranes unaided
• IAAH diffuses across membranes
• Auxin accumulates in more alkaline compartments

Cytosol Chloroplast 2/3 of IAA 1/3 of
IAA IAA is metabolized by conjugation or
conjugation and by nondecarboxylative
catabolism nondecarboxylation do not occur
in chloroplast
20
Auxin transport
Method for measuring polar auxin transport
Polar transport requires energy and is gravity
independent
Donor
(A)
(B)
Receiver
Donor
(B)
(A)
Auxin moves from apical to basal end
(basipetally) Unidirectional transport polar
transport
Receiver
21
A chemiosmotic model to explain polar auxin
transport
Mitchell, 1961 Chemiosmotic Theory chemical
potential across the membrane can provide energy
for ATP synthesis
Proton motive force (PMF) or ?p ?E ?pH
?E Transmembrane electric potential ?pH pH
difference across membrane
Auxin influx driven by PMF Auxin efflux driven by
?E
Influx entry/uptake of auxin into cells Efflux
loss of auxin from cells
For recap see Chapter 7
ATP synthase, ATPase
22
A chemiosmotic model to explain polar auxin
transport
1. IAA enters cell either passively (IAAH) or
actively (IAA-)
(AUX1)
2. Cell wall is maintained at acidic pH by
H-ATPase
3. IAA- predominates in cytosol
4. IAA- anions exit cell via auxin anion efflux
carriers (PIN proteins) concentrated at the basal
end of cell
Polarity of auxin transport is governed by efflux
step rather then the influx step.