Download Control of grape berry development

You can change this image to be
appropriate for your topic by inserting
an image in this space or use the
alternate title slide with lines.
Note: only one image should be used
and do not overlap the title text.
Enter your Business Unit or Flagship
name in the ribbon above the url.
Add collaborator logos in the white
space below the ribbon.
[delete instructions before use]
Control of grape berry development
Christine Böttcher, Silvia Dal Santo, Mario Pezzotti, Paul Boss, Christopher Davies
Davis, California 2013
CSIRO PI
Growth regulators and fruit ripening:
an old story
Ethylene in fruit development
Climacteric fruit
Non-climacteric fruit
tomato
apple
pear
blueberry
fig
grape
olive
strawberry
orange
*
RESPIRATION
*
RESPIRATION
ETHYLENE
ETHYLENE
TIME
TIME
The changing pattern of berry ripening
Climate change effects
•Earlier ripening
•Compressed ripening seasons
•Increased berry sugar - potential for increased wine alcohol levels
•Reduced flavour/aroma metabolite accumulation
PGRs possibly involved in fruit ripening
Abscisic acid (ABA)
Brassinosteroids
(BRs)
Ethylene
Castasterone
Auxins
Indole-3-acetic acid (IAA)
Indole-3-acetic acid
•elongation and division of cells
•organization of shoot and root architecture
•gravi- and phototropisms
•vascular development
Indole-3-acetic acid (IAA)
-Naphthalene acetic acid (NAA)
Auxin application can delay fruit ripening
Benzothiazole-2-oxyacetic acid (BTOA)
Davies et al., Plant Physiol. 1997
NAA delays grape berry ripening
Shiraz
2008/2009
Brix
pH units
Control (0.05% Tween 20)
*
Absorbance (520nm)
NAA (50 mg/L)
34 days post initial spray
Böttcher et al., 2011, AJGWR
*
Control
NAA
*
Days post initial spray
•Auxins delay ripening
in both climacteric
(bananas, tomatoes,
pears) and nonclimacteric fruit
(grapes, strawberries)
Headspace volatile and sensory analyses of
wines:
scale wines
Analysis of volatiles in headspace (SPME-GC-MS)
• 128 compounds identified
- 14 higher in ‘NAA wine’ – 10 of these were esters
- 5 higher in ‘Control wine’ – 4 aliphatic alcohols and linalool
• The differences were small, only one compound, more than two-fold different
Sensory analysis
• No differences detected by consumer panel
Böttcher et al., AJGWR (2010)
Experimental design
Pre-veraison Shiraz berries
Control v NAA treated berries
Ex planta
Three biological replicates
Time course sampled 3, 6, 12, 24, 48hr after treatment (deseeded)
PCA plot Nimblegen array Control v NAA
12h
6h
24h
3h
3h
48h
Genes significantly different at each time
point
1400
Number of genes
1200
1000
800
600
400
200
0
0
10
20
30
40
50
Time post-treatment (h)
60
Heat maps
NAA effects
Treatment sig.
downregulated
Treatment sig.
upregulated
Allocation by function
Lipid Generation Cell Wall Metabolism
Developmental
Metabolic of Energy
2%
Process
Process
2%
3%
Secondary
3%
Metabolic
Process
Unknown Protein
4%
14%
Response to Stress
4%
Cellular Amino Acids
and Derivative
Metabolic Process
5%
Pentatricopeptide
(PPR) repeatcontaining protein
1%
Transport
9%
Response to
Hormone Stimulus
5%
No Hit
8%
Cellular
Homeostasis
6%
Carbohydrate
Metabolic Process
6%
Transcription
Factor Activity
7%
Cellular
Process
7%
DNA/RNA
Metabolic
Process
8%
Signal
Transduction
7%
A little more detail
Hormone metabolism/signalling (auxin, ethylene, BR, ABA, JA, CK)
- indicates the a high degree of crosstalk between pathways
Transcription factors/protein fate
Cell wall metabolism
- down-regulation of pectin catabolic genes e.g. PG, PL, PME
Secondary metabolism
- potential to alter, phenyl propanoids, terpenoids
Three groups of gene ‘classically’
upregulated by auxins
SAUR – small auxin up-regulated proteins - function?
IAA/Aux - interact with ARFs and are negative regulators
of ARF action
GH3 enzymes - acyl amido synthetases, conjugate hormones
e.g. IAA, JA
Decline in IAA in grape berries important
FW
nmol/g
nmol/g FW
Cabernet Sauvignon
2004/2005
**
Weeks postflowering
• What controls the decrease in IAA levels prior to ripening?
Böttcher et al., 2010, J. Exp. Bot.
Metabolic fate of IAA: GH3 proteins
IBA-synthase
GH3-like proteins
IBA
ß-oxidation
?
MeIAA
amido hydrolases
indole-3-acetic
acid (IAA)
IAMT1
UGT84B1 hydrolase
IAA-glucose
IAA-amino acid
conjugates
?
?
degradation
IAA-proteins
•six indole-3-acetic acid-amido synthetases in grape
storage
Proposed reaction mechanism of GH3 proteins
+ ATP
+
PPi
IAA
AMP
Asp
IAA-Asp
GH3-like proteins (indole-3-acetic acid-amido synthetases)
first identified in soybean, found in mosses, gymnosperms, angiosperms
involved in storage or inactivation of IAA depending on the amino acid
substrate
Grapevine GH3 family
*
*
**
6
5
3
1
2
* functionally characterized
unknown function (4substituted benzoates)
**
**
*
*
*
*
*
IAA-amido synthetases
4
*
Böttcher et al., 2011, J. Exp. Bot.
jasmonic acid-amido synthetases
Expression of GH3 genes in grape berries
Copy number
*
*
*
*
Weeks post flowering
Böttcher et al., 2010, 2011, J. Exp. Bot.
*
*
Tissue-specific expression of GH3 genes
Copy number
Cabernet Sauvignon
2010/2011
Weeks postflowering
R: root T: tendril S: stem F: flower SL: small leaf ML: medium leaf LL: large leaf
unpublished
IAA-Asp accumulates in flowers and ripening
berries
IAA
IAA-Asp
Cabernet Sauvignon
2004/2005
pmol/g FW
pmol/g FW
•No other IAAconjugates detected
by LC-MS scan
*
Weeks postflowering
do GH3 proteins control IAA levels in berries?
Böttcher et al., 2010, J Exp Bot
Per gram
IAA and IAA-Asp in developing tomato fruit
IAA
pmol/g FW
Mature green
Turning
IAA-Asp
Light red
Ripe red
Böttcher et al., 2010, J. Exp. Bot.
Different auxins have different effects on Shiraz berry
development
60
Control
NAA
IAA
BTOA
25
20
anthocyanin level
(A 520nm) g FW-1
TSS (degrees Brix)
30
15
10
5
Sugar
0
0
20
40
60
80
100
days post initial spray
Control
NAA
IAA
BTOA
50
40
30
20
10
0
Colour
0
20
40
60
80
100
days post initial spray
average berry weight (g)
1.8
Control
NAA
IAA
BTOA
1.6
1.4
1.2
1.0
0.8
0.6
Shiraz
2009/2010
Weight
0.4
0
20
40
60
days post initial spray
Böttcher et al., 2011, J. Exp. Bot.
80
100
Transcriptional response of GH3s to PGRs
Gene
GH3-1
Auxins ABA Ethephon
upregulated
GH3-2
downregulated
GH3-3
GH3-4
GH3-5
GH3-6
no response
Exogenous IAA is inactivated by conjugation
IAA
Control
Copy number
pmol/g FW
IAA
GH3-2
IAA-Asp
Shiraz
2009/2010
Days post initial spray
Böttcher et al., 2011, J. Exp. Bot.
Substrate preferences of GH3-1 and GH3-2
V(nmol IAA-Asp min-1 mg protein-1)
Enzyme Substrate
GH3-1
IAA
NAA
BTOA
GH3-1
GH3-2
[IAA] (μM)
Böttcher et al., 2011, J. Exp. Bot.
GH3-2
Km
(µM)
10.4 ±1.2
190.3 26.2
n/a
Vmax
kcat
kcat/ Km
-1
-1
-1
(nmol min mg ) (min ) (M-1s-1)
14.3
5.2
22848
454.4
n/a
n/a
n/a
207.3 4.3
75.2 3.1
IAA
28.4 4.7
473.1 15.7
32.6
19148
NAA
554.3 92.2
435.9 27.4
30.1
904
BTOA
565.2 160
0.2 0.02
0.01
0.41
substrate preferences of GH3 proteins
and variations in the effect
of different auxins on grape berry ripening
are correlated (linked?)
Different auxins have different effects on Shiraz berry
development
60
Control
NAA
IAA
BTOA
25
20
anthocyanin level
(A 520nm) g FW-1
TSS (degrees Brix)
30
15
10
5
Sugar
0
0
20
40
60
80
100
days post initial spray
Control
NAA
IAA
BTOA
50
40
30
20
10
0
Colour
0
20
40
60
80
100
days post initial spray
average berry weight (g)
1.8
Control
NAA
IAA
BTOA
1.6
1.4
1.2
1.0
0.8
0.6
Shiraz
2009/2010
Weight
0.4
0
20
40
60
days post initial spray
Böttcher et al., 2011, J. Exp. Bot.
80
100
In vitro activity of the remaining five GH3s
Peat et al., 2012, Plant Cell
Protein
Preferred amino acid
substrate
GH3-1
Asp, (Trp)
GH3-2
Asp, (Trp)
GH3-3
Asp, (Trp, Met)
GH3-4
Glu (Gly, Met)
GH3-5
Asp (Gly, Ala)
GH3-6
Glu (Trp, Met)
Chemical inhibition
– another tool to study GH3 function
+
+
ATP
PPi
IAA
AMP
Asp
X = CO
IAA-Asp
Adenosine-5‘-[2-(1H-indol-3-yl)acetyl]phosphate
X = CH2 Adenosine-5‘-[2-(1H-indol-3-yl)ethyl]phosphate (AIEP)
Böttcher et al., 2012, PLoS ONE
Crystallisation of a GH3 protein
GH3 inhibitor
– a stable reaction intermediate
Vv GH3.1
C-domain
Conformational change of GH3 enzymes
during catalysis
Westfall et al., J. Biol. Chem. (2013) doi/10.1074/jbc.R113.475160
IAA
Binding of
different auxins
NAA
BTOA
Peat et al., 2012 Plant Cell
Page 35
Different auxins have different effects on Shiraz berry
development
60
Control
NAA
IAA
BTOA
25
20
anthocyanin level
(A 520nm) g FW-1
TSS (degrees Brix)
30
15
10
5
Sugar
0
0
20
40
60
80
100
days post initial spray
Control
NAA
IAA
BTOA
50
40
30
20
10
0
Colour
0
20
40
60
80
100
days post initial spray
average berry weight (g)
1.8
Control
NAA
IAA
BTOA
1.6
1.4
1.2
1.0
0.8
0.6
Shiraz
2009/2010
Weight
0.4
0
20
40
60
days post initial spray
Böttcher et al., 2011, J. Exp. Bot.
80
100
CEPA treatment early in berry development
delays ripening
biphasic response to ethylene in berries
17 days pre-veraison
Anthocyanin level (OD 520nm)
Anthocyanin level (OD 520nm)
7 days pre-veraison
25
Control
CEPA
20
15
10
5
0
0
10
20
30
40
50
60
12
10
Control
CEPA
8
6
4
2
0
0
Days post-spray
Bottcher et al., 2013, FPB
10
20
30
40
Days post-spray
Shiraz 2007/2008
50
60
In summary
At least five endogenous PGRs (and a
number of synthetic PGRs) can influence
ripening
•Cytokinins, auxins, ethylene retard
ripening
•ABA, ethylene, BRs advance ripening
In summary contd.
Indole-3-acetic acid-amido synthetases (GH3’s)
•may have an important role in controlling fruit ripening
Similarities between climacteric and non-climacteric fruit
•e.g. ethylene has a role in non-climacteric fruit ripening
•auxins ability to delay ripening
•role of GH3’s
Benefits to the wine industry
Changing the harvest season by advancing or delaying ripening
•mitigate harvest season compression due to climate change
(lengthen the harvest period)
•Improved winery intake scheduling - reducing costs
•allow fruit to be harvested at optimal time – improved quality
•manipulate fruit composition by direct effects, by delaying or
advancing ripening to cooler or warmer times of the season
•increased synchronicity of ripening
•reduced fruit wastage
Page 40
PGRs during grape berry development
PGRs and ripening
GA
Katie Harvey
Crista Burbidge
Rob Keyzers
Eric Dennis
Sue Maffei
Emily Nicholson
Ciaran Forde
Thank you
Tom Peat (CMSE)
Janet Newman (CMSE)
Grant Booker
Steven Polyak
PLANT INDUSTRY