On the move to field (5 min)
Lukáš Spíchal
1
1,2
Laboratory of Growth Regulators, IEB AS CR & UP Olomouc, Czech Republic
2
Centre of the Region Haná for Biotechnological and Agricultural Research,
Olomouc, Czech Republic
Acknowledgements
Laboratory of Growth Regulators, IEB AS CR & UP Olomouc :
Miroslav Strnad
Marek Zatloukal
Lucie Szüčová
Václav Mik
Karel Doležal
Markéta Gemrotová
Jana Klásková
Jaroslav Nisler
Jiří Grúz
Ondřej Novák
Jaromír Mikulík
Radoslav Koprna
boss
organic synthesis
biochemistry/biology
analytical chemistry
field experiments
Department of Biochemistry, PřF UP Olomouc:
Petr Galuszka
David Kopečný
Laboratory of Genome Integrity, UP Olomouc & BioPatterns, Ltd., Olomouc:
Martin Mistrík
Institute of Biology/Applied Genetics, FU Berlin:
Tomáš Werner
Thomas Schmülling
Grant Support: MSM 6198959216, LC06034, ED0007/01/01, GA522/08/0920
Development of CK-derived PGRs
TARGETED SYNTHESIS
Organic synthesis
Testing in CK bioassays
SAR
Testing of toxicity
Interaction with CK metabolism/perception
In vivo applications (temporal/spatial specific)
Phenotype
Gene profiling
Hormone levels
Testing of toxicity
In vivo applications in field conditions
What to change to gain…
R2
R1
H
H
C
C
C
H
N
N
H
N
H
C
C
N
Spíchal et al., 2009
Nisler et al., 2010
C N
C H
R1
C N
H
BA
N
H
N
C
C H H
H
C
H
H
N
N
N
N
N
C
NH
Cytokinin
antagonist
NH
CKX inhibitor
NH
BA
N
N
R2
N
N
H
Zatloukal et al., 2008
Spíchal et al., submitted
Development of plant growth regulators at Laboratory of Growth Regulators
(http://rustreg.upol.cz/)
Inhibitors of cytokinin signalling (PI-55)
Inhibitors of cytokinin degradation (INCYDE)
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
INCYDE
INCYDE
INhibitor of CYtokinin DEgradation
INhibitor of CYtokinin oxidase/DEhydrogenase
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
% of control
winter rapeseed 2009-2010
160
140
120
100
80
60
40
20
0
10 µM
50 µM
winter rapeseed 2010-2011
10% increase in field yeild
INCYDE
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 types of application
different phenological phases
different doses
INCYDE negatively affects Verticillium longisporum proliferation
control
A
Col-0 +
10µM INCYDE
infected
E
B
C
90
Col-0 + DMSO
Col-0 + 10 µM INCYDE
80
leaf area in cm²
Col-0 +
DMSO
28dpi
D
70
60
50
40
**
30
20
10
0
control
G
14
12
Col-0 + DMSO necrotic
Col-0 + INCYDE necrotic
10
8
Col-0 + DMSO chlorotic
Col-0 + INCYDE
chlorotic
6
4
***
2
0
control
45.00
40.00
infected
***
pg fungal DNA/108 actin 28dpi
chlorotic & necrotic leaves / plant
28dpi
F
***
infected
Col-0 + DMSO
Col-0 + 10 µM INCYDE
35.00
30.00
25.00
20.00
15.00
**
10.00
5.00
n.d.
n.d.
0.00
Col-0 +
DMSO
Col-0 +
INCYDE
Col-0 inf. + Col-0 inf. +
DMSO
INCYDE
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
PAL
trans-cinnamic acid
hydroxycinnamic acids (HCA)
Deikmann et al., 1995
INCYDE improves functional properties of edible plants
TPC - phenolic content
ORAC - radical scavenging
HBA - hydroxybenzoic acids
HCA - hydroxycinnamic acids
Grúz et al., unpublished
INCYDE is not cyto- and genotoxic
Cytotoxicity:
- epithelial and endothelial fibroblasts:
NEGATIVE
In vivo oral toxicity:
MTD > 400 mg/kg
Pharmacokinetics - elimination half life ~ 2 h
Genotoxicity:
- Ames test - NEGATIVE
- DNA damage - NEGATIVE
Conclusion
In the rigth time the right dose of INCYDE application
has positive effect on quantitative traits of plants
(increased size and number of flowers, fruits and seeds; increased biomass)
improves functional
properties of edible plants
protects against biotic stress - strongly reduces symptom
development and negatively affects Verticillium proliferation
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., 2004
Higuchi et al., 2004
Inhibitors of cytokinin signalling – PI-55, LGR-991
PI-55 reduces binding of the natural ligand to cytokinin receptors
PI-55 stimulates root branching and increases number of lateral roots
PI-55 accelaretes seed germination
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.
Anti-senescent activity and post-harvest storage
24 h pulsing + 10 days
T-2 (50 µM)
T-4 (50 µM)
Control (DMSO)
Shimon Meir, Volcani Center, Israel
New cotton defoliants derived from cytokinins
• Dropp 4 SC, Free Fall 50 WP,
Leafless 4 F, Ginstar 1.5 EC
contain thidiazuron as active compound
• GROSSMANN, K. (1990) Induction of Leaf Abscission in
Cotton Is a Common Effect of Urea-and Adenine-Type
Cytokinins. Plant Physiol. 234-237.
Defoliation of cotton leaves
100
90
Defoliation (%)
80
control
TDZ (250 µM)
control
LGR-951 (500 µM)
LGR-2210 (500)
70
LGR-1042 (500)
60
LGR-951 (500)
50
LGR-950 (500)
40
TDZ (250)
30
TDZ (500)
20
10
0
0
2
4
6
8
10
12
Days
Berlin 2009