Hormonal control of leaf expansion
in Arabidopsis
Christopher P. Keller
Minot State University
Minot, North Dakota, USA
•
50,000 people
•
(north) centrally located in
the great plains of North
American
•
80 km from the Canadian
border
Minot State University
• state-owned /supported university
• Master’s medium by Carnegie
classification
• 3,500 total enrolment
• students primarily from northwestern North Dakota but 25% are
from out-of-state
• 365 foreign students (31 countries)
“Auxin”
the first hormone
Frits Went (1926)
“Auxin”
the first hormone
4-chloroindole-3-acetic acid
2-phenylacetic acid
IAA
Indole-3-acetic acid
(ubiquitous and essential)
indole-3-butyric acid
IAA synthesis
AUX1 uptake transporter
Cell-to-cell
IAA transport in the
phloem parenchyma
PIN efflux transporter
Morphogenic control by IAA
• apical dominance
• phototropism
• lateral root initiation
• gravitropism
Is IAA also a morphogenic controller of leaf development?
?
Is IAA also a morphogenic controller of leaf development?
We now know…. that IAA accumulation initiates leaf primordium at the
margins of the shoot apical meristem.
(Reinhardt et al. [2003] Nature 426:255-260; Smith et al. [2006] PNAS 1301-1306)
?
Is IAA also a morphogenic controller of leaf development?
We also now know…. that IAA is involved in
controlling vascular development in very young
leaves.
(Aloni et al., [2003] Planta 216: 841-853)
?
Is IAA also a morphogenic controller of leaf development?
But 25 years ago…. IAA was thought to be of limited importance to leaf growth
the available data "fit in with the
view that auxin causes elongation of
leaf veins, while the growth of the
mesophyll depends on other factors"
?
(Went and Thimman [1937] Phytohormones.
Macmillan, New York)
H. Barbier-Brygoo and colleagues:
(1989) PNAS 86: 891-896
(1991) Plant J. 1(1): 83-93
Intracellular
microelectrode
Protoplast preparation
• Treatment with IAA (or other auxins) alters tobacco protoplast membrane potential
• This auxin effect could be blocked by antibodies raised to Auxin Binding Protein 1
(ABP1) – first evidence that ABP1 functions as a hormone receptor
Tobacco (Nicotiana tabacum)
From: Keller CP, Van Volkenburgh E. (1997) Plant Physiol. 113: 603-610
progress of the
cell-division/cell-expansion transition
Leaf
primordium
cell
expansion
growth
cell division
growth
leaf
≤ 20%
of final size
Young tobacco leaf transformed with ABP1
-tetracycline inducible promoter construct
Jones et al., (1998) Science
282: 1114-117
ABP1 modulates the tobacco leafstrip curvature response
auxin
auxin + tetracycline
If auxin induces growth of excised leaf strips, why had
Avery and Went and Thimman found no such effect in
intact plants?
the available data "fit in with the
view that auxin causes elongation of
leaf veins, while the growth of the
mesophyll depends on other factors"
?
(Went and Thimman [1937] Phytohormones.
Macmillan, New York)
Blade length 40-50mm
10-12 day old
Common bean
seedling
Common Bean (Phaseolus vulgaris)
leaf length (mm)
Exogenous 1mM IAA applied daily to leaves
slows growth (monofoliate midrib length).
100
100
100
90
90
90
80
80
80
70
70
70
60
60
60
50
50
50
40
40
40
30
30
30
20
20
20
A
10
B
10
0
0
0
0
1
2
3
4
5
6
7
C
10
0
1
2
3
4
5
6
7
days following treatment
0
1
2
3
4
5
6
7
leaf elongation (treated minus untreated) (mm)
6
4
2
0
-2
-4
-6
-8
A
-10
-12
0.001 0.01
0
0.1
1
10
100
1000 10000
leaf elongation (treated minus untreated) (mm)
Concentration (uM)
6
4
2
0
-2
-4
-6
-8
B
-10
-12
0.001 0.01
0
0.1
1
10
100
Concentration (uM)
1000 10000
AUX1 uptake transporter
XX
XX
NPA
(auxin efflux transporter inhibitor)
Cell-to-cell
IAA transport in the
phloem parenchyma
PIN efflux transporter
NPA
(in lanolin)
leaf length (mm)
120
Lanolin control
- treated leaf
100
80
Lanolin control
- untreated
leaf
NPA - treated
leaf
60
40
20
NPA untreated leaf
0
0
2
4
6
8
10 12 14 16 18 20
days following treatment
NPA treatment of leaf petioles results in smaller leaves
3500
4000
*
base
*
3500
3000
Cell area (µm2)
4000
mid-leaf
2500
2000
3000
3000
2500
2500
*
2000
tip
3500
*
2000
1500
1000
500
0
0
2
4
6
8
1500
1500
1000
1000
500
500
0
0
0
2
4
6
8
0
2
4
Days following treatment
NPA treated leaf has smaller cells
6
8
Auxin quantification - Cohen lab, U of Minnesota
-freeze at -80, transport on dry ice
-add [13C6]IAA internal standard
-grind/extract with isopropanol
-add 3H-IAA
-amino ion exchange column, elute
-HPLC
-pool radioactivity
-rotoevaporate
-methylate with diazomethane
-analyze with GC-MS using the ion peaks from the
internal standard for quantification
Days after
application
IAA (auxin)
concentration (ng/g)
# of leaf
pairs (n)
Significance
(paired t-test)
treated
untreated
1
53.2 +/- 17.2
29.3 +/- 14.4
4
0.025
treated
untreated
3
52.5 +/- 11.6
14.2 +/- 1.4
3
0.076
treated
untreated
6
8.9 +/- 0.7
6.3 +/- 0.78
4
0.030
Leaf
Application of NPA to petioles of expanding leaves elevates endogenous IAA
Opposite effects of IAA:
Inhibits leaf growth in intact plants
leaf length (mm)
120
Lanolin
control treated leaf
100
Lanolin
control untreated
leaf
NPA treated leaf
80
60
40
20
0
0
2
4
6
8
10 12 14 16 18 20
days following treatment
Induces growth in excised leaf strips?
Why?
NPA untreated
leaf
Three hypotheses:
1) Wound response
Microarray data suggests several IAA response genes are down regulated by
wounding.
Perhaps wounding reverses tissue sensitivity to IAA
wound insensitive
signal transduction
elements
IAA
wounding
wound sensitive
signal transduction
elements
increased
growth
decreased
growth
Three hypotheses:
) Detachment effect
Root derived hormones are needed for full leaf development.
Perhaps excision deprives leaf tissues of a non-IAA growth effecter that
modulates IAA control of leaf growth.
Three hypotheses:
3) Optimal concentration effect
Wounding (by chewing insects) in tobacco leaves has been shown to collapse
leaf IAA content (inducing transcription IAA suppressed defense genes) .
Perhaps in intact tissues IAA levels are optimal for growth induction and in
excised tissues IAA levels are suboptimal.
Intact leaf
growth
Excised
leaf
strip
Endogenous IAA concentration
The study system: 10-14 day old soil-grown Arabidopsis
first two true leaves 2.7-3.3 mm in diameter and rapidly expanding
excised
strips
intact
attached
Treatments: 24 hours
exposure to IAA at a range of
concentrations
wounded
attached
detached
intact
wounded
detached
Treatments:
(24 hrs+/- IAA)
Growth Response Predictions:
Wound Response Hypothesis
Detachment Effect Hypothesis
+
+
_
_
+
_
excised strips
intact attached
wounded attach
_
+
detached intact
+
wounded detached
+
excised strips
IAA treatment produces
epinastic (downward)
curvature regardless of
treatment
control
IAA
intact attached
control
IAA
wounded attach
control
IAA
detached intact
wounded detached
control
control
IAA
IAA
20
wounded attached
15
10
5
0
0.1
1
10
100
1000
intact attached
detached
intact
Difference in curvature area after 24h
(degrees / mm)
Concentration (µM)
50
40
30
20
10
0
wounded detached
-10
0.1
1
10
Concentration (µM)
100
1000
Difference in curvature area after 24h
(area lost to curvature as% of total area)
25
50
40
30
20
10
0
-10
0.1
1
10
100
1000
100
1000
Concentration (µM)
Difference in curvature area after 24h
(IAA minus contol, degrees / mm)
Difference in curvature area after 24h
(IAA minus contol, degrees / mm)
excised
strips
30
35
30
25
20
15
10
5
0
-5
0.1
1
10
Concentration (µM)
The adaxial (upper) surface is more sensitive to IAA-induced growth increase while
the abaxial (lower) surface is more sensitive to IAA-induced growth inhibition.
Control leaf expansion after 24 h
(%of initial)
control leaf expansion
(% of initial area)
100
80
60
40
20
0
differential relative growth
IAA minus control
(% of initial area)
Difference in relative growth after 24 h
IAA minus control (% of initial area)
high light
low light
excised strips
dark
25
20
high light
15
10
low light
5
dark
0
-5
0.1
1
10
100
1000
IAA concentration (µM)
IAA induced increased growth (expansion) of excised strips
but sensitivity was increased under stressful (high light) conditions
Control leaf expansion after 24 h
(%of initial)
control leaf expansion
(% of initial area)
100
80
60
40
20
detached intact
0
differential relative growth
IAA minus control
(% of initial area)
Difference in relative growth after 24 h
IAA minus control (% of initial area)
high light
low light
abaxial
10
abaxial
0
-10
high light
-20
-30
low light
-40
0.1
1
10
100
1000
IAA concentration (µM)
IAA inhibited expansion of intact detached leaves
but sensitivity was decreased under stressful (high light) conditions
Control leaf expansion after 24 h
(%of initial)
Control leaf expansion (% of initial area)
100
80
60
40
20
Intact
Wounded attached
Difference in relative growth after 24 h
IAA minus control (% of initial area)
0
10
0
-10
-20
-30
-40
-50
0.1
1
10
100
1000
IAA concentration (µM)
Differential relative growth
IAA minus control (% of initial area)
IAA inhibited intact attached leaf expansion
but wounding (without detachment ) reduced sensitivity
Control leaf expansion after 24 h
(%of initial)
control leaf expansion
(% of initial area)
100
80
60
40
wounded detached
20
0
differential relative growth
IAA minus control
(% of initial area)
Difference in relative growth after 24 h
IAA minus control (% of initial area)
wounded detached
35
30
25
20
15
10
5
0
-5
-10
0.1
1
10
100
1000
IAA concentration (µM)
IAA induced increased expansion of wounded detached leaves
IAA content (ng/g fresh wt.)
Endogenous IAA content after 24 hours
80
A
A
A
AB
AB
B
Approximately
0.5 µM
60
40
20
0
Control
Wounded
Detached
high Light
Detached
low light
Strips high
light
Strips low
light
Wounding and detachment were found to have no significant
effect on IAA endogenous content (which is remarkably low
relative to the effective exogenous concentrations)
Three hypotheses:
3) Optimal concentration effect
Wounding (by chewing insects) in tobacco leaves has been shown to collapse
leaf IAA content (inducing transcription IAA suppressed defense genes) .
Perhaps in intact tissues IAA levels are optimal for growth induction and in
excised tissues IAA levels are suboptimal.
Intact leaf
growth
Excised
leaf
strip
Endogenous IAA concentration
Conclusion:
IAA-induced increased growth (regardless of concentration) only occurred in
excised strips and detached wounded leaves.
Thus, IAA-induced growth increase results from a wound response that requires:
(1) substantial wounding (simple leaf excision is not enough as the growth of
intact detached leaves is inhibited by IAA) and
(2) detachment from the plant (because the growth of wounded attached leaves
is also inhibited by IAA) .
A model:
root derived factor
decreased
cell expansion
IAA
wound-induced factors
increased
cell expansion
root derived factor
An obvious next question:
root derived factor
?
decreased
cell expansion
IAA
wound-induced factors
increased
cell expansion
root derived factor
?
root derived growth effecter?
• mRNAs or microRNAs?
•cytokinins?
• strigolactones?
• abscisic acid?
root derived growth effecter?
• mRNAs or microRNAs?
Long distance transfer of mRNAs and microRNA in the phloem sap is
now well established in plants but the expanding 1st and 2nd leaves as
used in our experiments have likely already passed though the sink to
source transition.
root derived growth effecter?
• cytokinins?
Conditional cytokinin-deficient Arabidopsis transformants have impaired leaf
expansion due to
root derived growth effecter?
GR24
Synthetic strigolactone
• strigolactones?
Increase in detached leaf area
increase in area (% of initial)
increase in area (% of initial)
Results of treatment of detached leaves with GR24 does not suggest a role for
strigolactones in leaf growth inhibition
Increase in detached leaf area
100
120
100
80
60
40
20
0
Control
GR24
80
60
40
20
0
IAA
IAA + GR24
root derived growth effecter?
• Abscisic acid (ABA)?
- the plant stress hormone
- growth inhibitor
synthesis also in leaves
transport in the xylem stream
synthesis in roots
Effect of ABA on IAA-induced leaf strip growth
Effect of IAA on leaf strip growth
70
60
50
40
30
20
10
0
-10
-20
-30
-40
control
control plus IAA
difference
(treatment control)
increase in area at 24 h (% of
initial)
increase in area at 24h (% of initial)
Excised Leaf Strip
70
60
50
40
30
20
10
0
-10
-20
-30
-40
Difference
(treatment –
Control)
IAA
IAA and ABA
IAA VS IAA And ABA
IIAA VS Control
ABA inhibits expansion of Arabidopsis leaf strips (+/- IAA)
increase in area (% of initial)
increase in area (% of initial)
abamineSG:
ABA synthesis inhibitor
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
1
Control
2
abamineSG
1=control, 2=abamineSG
1
IAA
2
IAA + abamine
1=IAA, 2=IAA and abamineSG
Growth of detached leaves is enhanced by abamineSG and IAAinduced growth inhibition is blocked by abamineSG
increased area (% of initial)
Wild type Arabidopsis
120
100
91.07
73.75
80
60
40
20
0
C
1
control
2
IAA
increased area (% of initial)
Intact Attached Plant
ABA deficient mutant (cs5736)
120
100
80
60
40
20
0
1
control
2
IAA
Growth of attached leaves of an ABA synthesis deficient mutant is not inhibited by IAA
Wild type
100
81.66
90
80
61.42
70
60
50
40
30
20
increase in area at (% of initial)
increase in area (% of initial)
Detached Leaf
ABA deficient mutant (cs5736)
120
100
80
60
40
20
10
0
0
1
2
1
2
control
IAA
control
IAA
Treatment: 1=control, 2=IAA
treatment (1=control, 2=IµM 50IAA
Growth of detached leaves of the ABA synthesis deficient mutant is also not inhibited by IAA
root derived factor
(ABA)
Two possibilities:
A1: transport of IAA to
roots and induction of
increase root ABA
decreased leaf
cell expansion
A2: induction of increased ABA synthesis in
treated leaf
A
1) Increased IAA induces increased ABA
production in roots and in unwounded
leaves
IAA
wound-induced factors
?
B
increased leaf
cell expansion
root derived factor (ABA)
Or
root derived factor
(ABA)
A1:increased growth
sensitivity to root derived
ABA
decreased leaf
cell expansion
A2: increased growth sensitivity to ABA synthesized in the leaf
2) Increased ABA induces increased ABA
growth sensitivity in unwounded leaves
A
IAA
wound-induced factors
?
increased leaf
cell expansion
B
root derived factor (ABA)
Test of sensitivity hypothesis:
Pretreat 6 hours in either control solution or IAA then ABA for 24 hours
24 hours in ABA
80
increase in area (% of initial)
increase in area (% of initial)
pretreatment
70
60
50
40
30
20
10
0
1
Control
2
IAA
80
70
60
50
40
30
20
10
0
1
control to ABA
2
IAA to ABA
6 hour pretreatment with IAA does not increase sensitivity to ABA
Test of ABA induction
hypothesis:
excised
strips
intact
attached
Determination of ABA content
of variously treated samples
wounded
attached
detached
intact
wounded
detached
Test of ABA induction
hypothesis:
excised
strips
intact
attached
Determination of ABA content
of variously treated samples
To be completed with another visit to
the Cohen lab.
wounded
attached
detached
intact
wounded
detached
Acknowledgements
Minot State University :
Michael Evanoff
Josh Glaser
Morgan Grundstad
Jeremy Keith
Derek Lentz
Amanda Roise
Samuel Wagner
Jakob Zerr
University of Minnesota:
Jerry Cohen
Angella Culler
Lana Barkawi
This project is supported by grants from the
National Center for Research Resources
(P20RR016471) and the National Institute of
General Medical Sciences (P20 GM103442)
from the National Institutes of Health.