Cytokinin modifications for versatile Cytokinin modifications for versatile functions and assessment of their functions and assessment of their

biological activitybiological activity

Department of Department of Molecular BiologyMolecular Biology

Kateřina Václavíková, David Zalabák, Lucie Szüčová, Karel Doležal, Kateřina Václavíková, David Zalabák, Lucie Szüčová, Karel Doležal, Jaroslav Nisler, Lukáš Spíchal, Miroslav Strnad, Petr GaluszkaJaroslav Nisler, Lukáš Spíchal, Miroslav Strnad, Petr Galuszka

Department of Department of Growth Growth RegulatorsRegulators

Ondřej PlíhalOndřej Plíhal

Aromatic cytokinins for plant growth Aromatic cytokinins for plant growth and regenerationand regeneration

positive effects. positive effects. Important use in Important use in micropropagation techniques. Modification micropropagation techniques. Modification of growth, organ differentiation… BAP and of growth, organ differentiation… BAP and kinetin are usually preferred.kinetin are usually preferred.

negative effects. negative effects. Inhibitory effects on Inhibitory effects on normal root development. BAP itself normal root development. BAP itself seems to reduce mitotic activity in the root seems to reduce mitotic activity in the root meristem.meristem. Growth enhancement Growth enhancement

mediated by cytokininsmediated by cytokinins

How to reduce negative effects of How to reduce negative effects of cytokinins application?cytokinins application?

use of topolins. use of topolins. Topolins and their Topolins and their methoxy counterparts are in some methoxy counterparts are in some cases preferred over BAP.cases preferred over BAP.

use of synthetic derivatives. use of synthetic derivatives. Some substituents at N9 position Some substituents at N9 position proved to maintain significant cytokinin proved to maintain significant cytokinin activity. Modifications preserve activity activity. Modifications preserve activity (glycosylation impeded). Cytokinin (glycosylation impeded). Cytokinin derivatives N9-(2-tetrahydropyranyl) derivatives N9-(2-tetrahydropyranyl) and N9-(2-tetrahydrofuranyl) can be and N9-(2-tetrahydrofuranyl) can be used to promote plant development.used to promote plant development.

Arabidopsis treatmentsArabidopsis treatments

THP N9THP N9protectionprotection

In vitro In vitro cultivation experiments - maizecultivation experiments - maize

CONTROLCONTROL

THP N9THP N9protectionprotection

Co

mp

ou

nd

con

centratio

nC

om

po

un

d co

ncen

tration

In vitro In vitro cultivation experiments – maizecultivation experiments – maizeEffects on roots and leavesEffects on roots and leaves

reduced inhibition reduced inhibition effects. effects. N9 THP N9 THP protection facilitates protection facilitates normal root development.normal root development.

positive effects of N9 positive effects of N9 THP protection. THP protection. Positive Positive impact on overall organ impact on overall organ development.development.

CK

PP

PP

CHAS

E

H

D

CHASE

H

D

active HK dimer

YojN

RcsBcps::lacZ

β-galactosidaseexpression

Fluorescent assay- Quantification

KMI001KMI001rcsC, cps::lacZrcsC, cps::lacZ

And how about the cytokinin activity of And how about the cytokinin activity of N9 derivatives? N9 derivatives?

in vitroin vitro receptor receptor activations in activations in KMI001 cells. KMI001 cells. Precise Precise quantification of HK quantification of HK receptor activation receptor activation using using --galactosidase as a galactosidase as a marker gene.marker gene.

AHK vs ZmHK. AHK vs ZmHK. AHK3 and AHK3 and AHK4 available in pPIN-III AHK4 available in pPIN-III vector. ZmHK1 and ZmHK3a vector. ZmHK1 and ZmHK3a were cloned and tested in the were cloned and tested in the same expression vector. +/- same expression vector. +/- IPTG induction.IPTG induction.

Receptor testing - ArabidopsisReceptor testing - Arabidopsis

different ligand preferences. different ligand preferences. 3MeOBAP not active in AHK4.3MeOBAP not active in AHK4.

no activity of N9 derivatives.no activity of N9 derivatives.

Receptor testing - maizeReceptor testing - maize

maize vs Arabidopsis. maize vs Arabidopsis. Maize HK receptors are more Maize HK receptors are more sensitive to aromatic cytokinins sensitive to aromatic cytokinins than Arabidopsis HK.than Arabidopsis HK.

no activity of N9 derivatives.no activity of N9 derivatives.

Biological activity of N9 protected Biological activity of N9 protected cytokinins - SUMMcytokinins - SUMMAARYRY

active in active in in vitroin vitro cytokinin assays. cytokinin assays. Tobacco callus Tobacco callus assay and Amaranthus assay.assay and Amaranthus assay.

show comparable properties as unprotected parent show comparable properties as unprotected parent cytokinins on plant development. cytokinins on plant development. Positive cytokinin-like Positive cytokinin-like effects on plant development + reduced negative effects effects on plant development + reduced negative effects on root morphology.on root morphology.

no activity in HK receptor assaysno activity in HK receptor assays. Is there a . Is there a mechanism accountable for the apparent contradiction in mechanism accountable for the apparent contradiction in bioactivity vs ligand properties?bioactivity vs ligand properties?

Cytokinin content in maize roots after Cytokinin content in maize roots after treatment with 3MeOBAP and 3MeOBA9THPPtreatment with 3MeOBAP and 3MeOBA9THPP

T = 1 hT = 1 h T = 3 hT = 3 h

T = 24 hT = 24 h T = 72 hT = 72 h

*

*

*

*

??

Cytokinin content in maize leaves after Cytokinin content in maize leaves after treatment with 3MeOBAP and 3MeOBA9THPPtreatment with 3MeOBAP and 3MeOBA9THPP

T = 1 hT = 1 h T = 3 hT = 3 h

T = 24 hT = 24 h T = 72 hT = 72 h

*

*

**

??

Feeding experiments with radioactively Feeding experiments with radioactively labelled 3MeOBA9THPPlabelled 3MeOBA9THPP

…

RT (min)

Standardroot shoot root shoot

3-days application 7-days application

2-3 Adenine 2.66% 4.81% 1.93% 4.78%4-6 Adenosine, (OG)3OHBAP 7.56% 15.26% 5.05% 15.21%8-9 3MeOBAPR5MP, 3OHBAP, (OG)3OHBAP 1.80% 3.19% 0.68% 1.47%

11-12 3MeOBAP9G, 3OHBAPR 4.90% 2.71% 5.39% 0.90%18-19 3MeOBAP 2.44% 8.57% 2.22% 12.42%20-21 3MeOBAPR 1.75% 0.73% 0.43% 0.56%23-26 Unknown 8.34% 40.18% 8.02% 27.46%28-30 3MeOBA9THPP 70.56% 24.54% 76.28% 37.20%

The retention time (RT) of radioactivity The retention time (RT) of radioactivity detected after application of labeled detected after application of labeled 3MeOBA9THPP. 3MeOBA9THPP. Cytokinin standards eluted in Cytokinin standards eluted in corresponding RTs are listed together with level corresponding RTs are listed together with level of detected radioactivity.of detected radioactivity.

Expression profiles of genes involved in Expression profiles of genes involved in the cytokinin biosynthesisthe cytokinin biosynthesis

Expression profiles of genes involved in Expression profiles of genes involved in the the cytokinin biosynthesiscytokinin biosynthesis

CONCLUSIONSCONCLUSIONS

Some cytokinin derivatives at N9 can be Some cytokinin derivatives at N9 can be metabolized in maize.metabolized in maize. THP modification at N9 significantly enhances THP modification at N9 significantly enhances acropetal transport of a given cytokinin.acropetal transport of a given cytokinin.

protection impedes the accumulation of non-active protection impedes the accumulation of non-active cytokinin glucoside forms in roots.cytokinin glucoside forms in roots.

gradual release of the active base and its transport gradual release of the active base and its transport offer better distribution of the active cytokinin form.offer better distribution of the active cytokinin form.

amounts of naturally occurring isoprenoid amounts of naturally occurring isoprenoid cytokinins in different plant tissues are not strongly cytokinins in different plant tissues are not strongly affected when plants are treated with protected affected when plants are treated with protected cytokinin.cytokinin.

Thank you for your attention!Thank you for your attention!