Modulating Stomatal Activity For Water Use Efficiency And Stress Tolerance
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Transcript of Modulating Stomatal Activity For Water Use Efficiency And Stress Tolerance
"Modulating Stomatal Activity for
Water Use Efficiency and
Stress Tolerance"
CHIARA TONELLIUniversità degli Studi di Milano
Department of Biosciences
Enhancing water use efficiency & drought tolerance
Low water use efficiency
Low drought tolerance
Transpirational
water loss
Reduced transpirational
water loss
Enhanced water use efficiency
Enhanced drought tolerance
The «key» gene
The right key: a stomata (gate)-specific gene
Cominelli-Galbiati-Tonelli, Curr. Biol., 2005
Galbiati-Tonelli, Plant J., 2008
MYB60
AtMYB60 transcript accumulates in guard cells
under conditions promoting stomatal opening
• Dark
• Desiccation
• ABA
• Light (blue)
function?
Stomatal
closureStomatal
opening
LB
-3bp
wild-type atmyb60-1
atmyb60-1 is a knock-out allele of AtMYB60
AtMYB60
TSB1
Whole leaf LCM-GC
WT mut WT mut
?
Sto
mata
l in
dex
0
10
20
30
40
Num
ber
of
sto
mata
per
mm
2
0
100
200
300
400
dark
white light 3h
white light 6h
Sto
mata
l apert
ure
(m
m)
wild-type atmyb60-10
1
2
3
4
3h 6hDark
wild-type
3h 6h
atmyb60-1
Dark
wild type
atmyb60-1
C60-1
C60-2
C60-3
0
1
2
3
4
Sto
mata
l apert
ure
(m
m)
A functional AtMYB60 gene is required for proper light-induced opening
0
20
40
60
80
100
0 2 4 8
atmyb60-1
Time after leaf removal (h)
wate
r lo
ss (
%)
wild type
60.5
wild-typeatmyb60-1
00
10 20 30 40
20
40
60
80
wild type
atmyb60-1
Drought treatment time (day)
soil
wate
r conte
nt
(%) 100
0
40
60
80
100
20
wild typeatmyb60-1
Drought treatment time (day)
0 8 16
rela
tive w
ate
r conte
nt
(%)
0 8 16
The atmyb60-1 mutation enhances dehydration avoidance responses
MYB60
• Dark
• Desiccation
• ABA
• Light (blue)Stomatal
closureStomatal
opening
target genes
RNAseq GC
WT vs atmyb60-1
386 134
Finding the downstream targets
-3
-2
-1
0
1
2
3
Fo
ldch
an
ge
MYB60 ver WT (measured in GC) GC versus leaves
PS. Light reactions
VLCFA synthesis
Lipid transfer proteins
Nitrate Reductase
Aspartate metabolism
Metal handling
Glucosinolate synthesis
Flavonoids
Jasmonate metabolism
Stress Biotic
Biotic receptors
PR proteins
Different
WRKY TFs
Pseudo ARR TFs
DNA
Chromatin structure
Protein synthesis
Protein degradation
DUF kinases
Calcium signalling
Transport
Transport
ABC transporters
Not assigned
C
B
FC FC
locus name myb60 GC / L paralogs
AT1G25450 KCS5 0,44 10,32
AT1G68530 KCS6 0,51 5,07 96
AT1G04220 KCS2 0,57 1,04 96
AT1G07720 KCS3 0,61 5,16 30
AT2G28630 KCS12 0,62 2,14 96
AT1G01120 KCS1 0,63 0,98 30, 96
AT4G14440 HCD1 0,62 3,32
AT2G38530 LTP2 0,37 0,07
AT5G59310 LTP4 0,37 1,41
AT2G47240 CER8 0,58 56,02
AT4G00360 CYP86A2 0,62 3,06 30
AT4G24510 CER2 0,33 9,96 30
AT1G01610 GPAT4 0,57 3,02 30
134 up 386 down
MYB60 MYB60
GO terms: from single genes to metabolic pathways
Glucosinolate genes
Lipid genes
Defence genes
Glusinolate metabolism
Lipid metabolism
Defence responses
MYB60
Glucosinolate genes
Lipid genes
Defence genes
Glusinolate metabolism
Lipid metabolism
Defence responses
GC-MS analysis of the Guard Cell lipidome
wild type atmyb60-1
Aleksandra Skirycz and Patrick Giavalisco
Max Planck Institute of Molecular Plant Physiology
The unusual suspects: oxylipins
0
10
20
30
40
50
60
70
80
90
DGDG OPDA/OPDA dnOPDA dinorOPDA MGDG OPDA/dnOPDA MGDG OPDA/OPDA OPDA
ng/
g d
ry w
eig
ht
(th
ou
san
d)
GC atmyb60-1
GC WT
Leaf atmyb60-1
Leaf WT
0
20
40
60
80
100
120
140
WT atmyb60-1
LOX2
Guard Cells
Mesophyll
0
0.5
1
1.5
WT atmyb60-1
LOX5
Mesophyll
1. Preferentially expressed in the mesophyll
0
0.5
1
1.5
2
2.5
WT atmyb60-1
LOX3
Guard Cells
Mesophyll
2. Equally expressed in GC and mesophyll cells
3. Preferentially expressed in GCs
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
WT atmyb60-1
Re
lati
ve e
xpre
ssio
n
LOX1
Guard Cells
Mesophyll
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
WT atmyb60-1
LOX4
Guard Cells
Mesophyll
00
01
01
02
02
03
03
WT atmyb60-1
LOX6
Guard Cells
Mesophyll
aos mutants, devoid of oxylipine,show fully open stomata
opr3 mutants accumulate oxylipine and show partially open stomata like myb60-1
One key for many crops?
Galbiati-Tonelli, BMC Plant Biol., 2011
Cominelli-Galbiati-Tonelli, BMC Plant Biol., 2011
tobacco tomato grape
Identification of the AtMYB60 “minimal promoter”:
246 bp are sufficient to drive guard cell expression
GUS
-16-1291 bp
GUS
GUS
GUS
-619 -262 2219 bp
GUS
-205
The AtMYB60 “minimal promoter” is enriched in DOF binding sites,
over-represented in promoter of guard cell expressed genes
Re-programming AtMYB60 promoter activity in GCs
pMYB60 GUS
pMYB60 GUS
DRE ABRE
pMYB60 GUS
GU
S r
ela
tive
exp
ressio
n
pMYB60 pMYB60
Rusconi, F. et al. J. Exp. Bot. 64, 3361–3371 (2013).
ABA/Drought
Control
Independent lines
Exploiting the AtMYB60 promoter in tomato
pMYB60 GUS
GU
S r
ela
tive
exp
ressio
n
100mM ABA
0 0.5 1 4 8Time (h)
pMYB60 GUS
rd29a
DRE ABRE100mM ABA
GU
S r
ela
tive
exp
ressio
n
pMYB60 GUS
control ABA
AtMYB60: from grape to Arabidopsis
AtMYB60
VvMYB60
(J.T. Matus)
Conserved function?
1. Promoter
2. Protein
MYB60 ideal Target for gene editing (tomato and grape)
Advantages
• Cell specific expression
• NO pleiotropic effects on growth and productivity
• Highly conserved sequence and function in dicots
• Reduced water loss
• Efficient water use
• Drought tolerance
Acknowledgments
Massimo Galbiati
Lucio Conti
• Alain VavasseurCommissariat à l’Energie Atomique, St Paul lez Durance, France
• Francois TardieuINRA, Montpellier, France.
• Rainer HedrichWürzburg University, Germany
• Aleksandra Skirycz and Patrick GiavaliscoMax Planck Institute of Molecular Plant Physiology
Collaborations
Funding agencies
Lab members
Eleonora Cominelli
Matteo Riboni
Giulia Castorina
Sara Castelletti
Alice Robustelli Test
Fabio Rusconi
Laura Simoni