on the move to field (5 min)

On the move to field (5 min)

Lukáš SpíchalLaboratory of Growth Regulators, IEB AS CR & UP Olomouc, Czech Republic

Centre of the Region Haná for Biotechnological and Agricultural Research, Olomouc, Czech Republic

1,2

1

2

Laboratory of Growth Regulators, IEB AS CR & UP Olomouc :Miroslav StrnadMarek ZatloukalLucie SzüčováVáclav MikKarel DoležalMarkéta GemrotováJana Klásková Jaroslav NislerJiří GrúzOndřej NovákJaromír MikulíkRadoslav Koprna

Department of Biochemistry, PřF UP Olomouc:Petr GaluszkaDavid Kopečný

Laboratory of Genome Integrity, UP Olomouc & BioPatterns, Ltd., Olomouc:Martin Mistrík

Institute of Biology/Applied Genetics, FU Berlin:Tomáš WernerThomas Schmülling

Acknowledgements

Grant Support: MSM 6198959216, LC06034, ED0007/01/01, GA522/08/0920

organic synthesis

biochemistry/biology

analytical chemistryfield experiments

boss

Development of CK-derived PGRs

Organic synthesis

Testing in CK bioassays

Interaction with CK metabolism/perception

In vivo applications (temporal/spatial specific)

In vivo applications in field conditions

SAR

PhenotypeGene profilingHormone levels

Testing of toxicity

Testing of toxicity

TAR

GE

TED

SY

NTH

ES

IS

What to change to gain…

NHN

NN

NH

BA

NHN

NN

NH

R1

R2

NH

NHN

NN

R1

R2

NCC

C

N

NC

C

N

NC

CC

CC

C

C

H

H

H

H

H

H

H

HH

H

H

BA

Cytokininantagonist

CKX inhibitor

Spíchal et al., 2009Nisler et al., 2010

Zatloukal et al., 2008Spíchal et al., submitted

Development of plant growth regulators at Laboratory of Growth Regulators(http://rustreg.upol.cz/)

Inhibitors of cytokinin degradation (INCYDE)

Inhibitors of cytokinin signalling (PI-55)

Inhibitors of cytokinin biosynthesis

Cotton defoliants

Stimulators of legume nodulation (N2-INCYDE)

Vigour enhancing compounds and anti-senescence activity

development of fluorescent cytokinins

development of cytokinin derivatives for affinity purification of CK binding proteins

Modulation of cytokinin degradation

Ashikari et al., 2005

Bartrina et al., 2011

Petr Galuszka – lecture

INCYDEINhibitor of CYtokinin DEgradation

INhibitor of CYtokinin oxidase/DEhydrogenase

INCYDE

INCYDE rescues WT phenotype of CKX overproducing plants

INCYDE action

INCYDE inhibits CKX activity √Can we phenocopy the ckx mutants by INCYDE application?

Targeted INCYDE application on Arabidopsis WT plants

- INCYDE + INCYDE

Targeted INCYDE application on Arabidopsis WT plants

INCYDE as a growth regulator for modulation of traits of crops

winter rapeseed 2009-2010

Branches p

er stem

Siliques

per branch

Total yiel

d

Biomass (

DW, shoot)

Biomass (

DW, root)

020406080

100120140160

10 µM50 µM

% o

f con

trol

INCYDE

winter rapeseed 2010-2011

10% increase in field yeild

Field trials

winter rapeseed 2009-2010, 2010-2011

winter wheat 2009-2010, 2010-2011

barley 2010, 2011

maize 2010, 2011

rice 2010, 2011

different regions

different phenological phasesdifferent doses

different types of application

control 28dpi infected

INCYDE negatively affects Verticillium longisporum proliferation

7dpi

14dp

i21

dpi

28dp

i7d

pi14

dpi

21dp

i28

dpi

0

10

20

30

40

50

60

70

80

control infected

leaf

are

a in

cm

²

control infected0

1

2

3

4

5

6

7

8

9

10

Col-0 + DMSO necrotic

Col-0 + INCYDE necrotic

Col-0 + DMSO chlorotic

Col-0 + INCYDE chlorotic

chlo

rotic

& n

ecro

tic le

aves

/ pl

ant

28dp

i

Col-0 +10µM INCYDE

Col-0 +DMSO

Col-0 + DMSOCol-0 + 10 µM INCYDE

A B

C D

E

F G

*** ***

*****

Col-0 + DMSO

Col-0 + INCYDE

Col-0 inf. + DMSO

Col-0 inf. + INCYDE

0.00

5.00

10.00

15.00

20.00

25.00

30.00

pg

fung

al D

NA/1

08 a

ctin

28d

pi

Col-0 + DMSOCol-0 + 10 µM INCYDE

**n.d. n.d.

T. Teichmann, unpublished

INCYDE improves functional properties of edible plants

Functional foods can be defined as those providing health benefits beyond basic nutrition and include whole, fortified, enriched or enhanced foods which have a potentially beneficial effect on health when consumed as part of a varied diet on a regular basis at effective levels (Hasler CM, 2000).

Shikimate pathway hydroxybenzoic acids (HBA)

L- Phe

trans-cinnamic acid

hydroxycinnamic acids (HCA)

PAL

Deikmann et al., 1995

INCYDE improves functional properties of edible plants

ORAC - radical scavengingTPC - phenolic content

Grúz et al., unpublished

HBA - hydroxybenzoic acids HCA - hydroxycinnamic acids

INCYDE is not cyto- and genotoxic

Cytotoxicity:

- epithelial and endothelial fibroblasts:NEGATIVE

In vivo oral toxicity:

MTD > 400 mg/kgPharmacokinetics - elimination half life ~ 2 h

Genotoxicity:

- Ames test - NEGATIVE- DNA damage - NEGATIVE

Conclusion

In the rigth time the right dose of INCYDE application

improves functional properties of edible plants

protects against biotic stress - strongly reduces symptom development and negatively affects Verticillium proliferation

has positive effect on quantitative traits of plants (increased size and number of flowers, fruits and seeds; increased biomass)

INCYDE is not cytotoxic, genotoxic and shows very high tolerance in oral toxicity in vivo studies

Inhibitors of cytokinin signalling

Riefler et al., 2006

Nishimura et al., 2004Higuchi et al., 2004

Inhibitors of cytokinin signalling – PI-55, LGR-991

PI-55 stimulates root branching and increases number of lateral roots

PI-55 accelaretes seed germination

PI-55 reduces binding of the natural ligand to cytokinin receptors

PI-55 antagonizes cytokinin effects in bioassays with different plant species

PI-55 protects development of seedlings grown on contaminated soils

Development of compounds stimulating nodulation and nitrogen fixation of legumes

Regulation of perception and metabolism of cytokinins, important plant hormones, was recently shown to be very important during the nodule formation of legumes (Gonzalez-Rizzo et al., 2006; Murray et al., 2007; Tirichine et al., 2007; Lohar et al., 2004). Preliminary data shows that application of the tested compounds to the roots of pea leads to increment in nodule number and subsequent higher rate of nitrogen fixation. Importantly, in comparison to classical cytokinins the applications of our substances has no negative effects on root and shoot growth and development.

24 h pulsing + 10 days

Control (DMSO)

Anti-senescent activity and post-harvest storage

T-2 (50 µM) T-4 (50 µM)

Shimon Meir, Volcani Center, Israel

New cotton defoliants derived from cytokinins

control

control

TDZ (250 µM)

LGR-951 (500 µM)

Defoliation of cotton leaves

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12

Days

Defo

liatio

n (%

) LGR-2210 (500)

LGR-1042 (500)

LGR-951 (500)

LGR-950 (500)

TDZ (250)

TDZ (500)

• Dropp 4 SC, Free Fall 50 WP, Leafless 4 F, Ginstar 1.5 EC

• GROSSMANN, K. (1990) Induction of Leaf Abscission in Cotton Is a Common Effect of Urea-and Adenine-Type Cytokinins. Plant Physiol. 234-237.

contain thidiazuron as active compound

Berlin 2009