"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

Closing the gate

H2O & O2CO2

The «key» gene

The right key: a stomata (gate)-specific gene

Cominelli-Galbiati-Tonelli, Curr. Biol., 2005

Galbiati-Tonelli, Plant J., 2008

GFP

AtMYB60 is expressed in guard cells

ACTIN2

AtMYB60

LCM-purified cells

Me GC

GFP

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

wild-type

atmyb60-1

“Whole-plant” effect of the atmyb60-1 mutation

H2OH2O

H2OH2O???

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

AtMYB60 GC response

AtMYB31

AtMYB30

AtMYB96

AtMYB94

lipid-/wax

metabolism

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

GC Laser Micro Dissection

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

pL

OX

6:G

US

Co

l-0

pL

OX

6:G

US

atm

yb

60

-1

1 mm 10 mm

1 mm 10 mm0.1 mm

pL

OX

4:G

US

Co

l-0

pL

OX

4:G

US

atm

yb

60

-1

aos mutants, devoid of oxylipine,show fully open stomata

opr3 mutants accumulate oxylipine and show partially open stomata like myb60-1

MYB60

LOXs

MYB60

LOXs

AtMYB60: a negative regulator of OPDA synthesis in GC

OPDA

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

rice

tobacco

microtom

tomato

J Exp Bot 2013 doi:10.1093/jxb/ert180

AtMYB60 promoter in crops

GUSGUS

A true GC-specif promoter

ABA dehydration

Downregulated by drought

A possible way out

AtMYB60

rd29AABA-down

ABA-up rd29A

AtMYB60

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

A GC- drought-inducible promoter (tobacco)

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

AtMYB60: from grape to Arabidopsis

ABA

VvMYB60pVvMYB60

2.2kb

controlpVvMYB60 GUS

AtMYB60: from grape to Arabidopsis

VvMYB60

Sto

mata

l apert

ure

(m

M)

VvMYB60pro GFP

0

1

2

3

4

5

6

7

8

9

10

GC mesophyll

Re

lati

ve V

vMY

B6

0 e

xpre

ssio

n

VvMYB60

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