1. Cytokinin modifications for versatile functions and assessment of their biological activity Department of Molecular Biology Kateřina Václavíková, David Zalabák, Lucie Szüčová, Karel Doležal, Jaroslav Nisler, Lukáš Spíchal, Miroslav Strnad, Petr Galuszka Department of Growth Regulators Ondřej Plíhal

2. Aromatic cytokinins for plant growth and regeneration positive effects. Important use in micropropagation techniques. Modification of growth, organ differentiation… BAP and kinetin are usually preferred.  positive effects. Important use in micropropagation techniques. Modification of growth, organ differentiation… BAP and kinetin are usually preferred.  negative effects. Inhibitory effects on normal root development. BAP itself seems to reduce mitotic activity in the root meristem. Growth enhancement mediated by cytokinins

3. How to reduce negative effects of cytokinins application?  use of topolins. Topolins and their methoxy counterparts are in some cases preferred over BAP.  use of synthetic derivatives. Some substituents at N9 position proved to maintain significant cytokinin activity. Modifications preserve activity (glycosylation impeded). Cytokinin derivatives N9-(2-tetrahydropyranyl) and N9-(2-tetrahydrofuranyl) can be used to promote plant development.

4. Arabidopsis treatments THP N9 protection

5. In vitro cultivation experiments - maize CONTROL THP N9 protection Compound concentration

6. In vitro cultivation experiments – maize Effects on roots and leaves  reduced inhibition effects. N9 THP protection facilitates normal root development.  positive effects of N9 THP protection. Positive impact on overall organ development.

7. CK PP PP CHASECHASE H D CHASECHASE H D active HK dimer YojN RcsB cps::lacZ β-galactosidase expression Fluorescent assay - Quantification KMI001  rcsC, cps::lacZ And how about the cytokinin activity of N9 derivatives?  in vitro receptor activations in KMI001 cells. Precise quantification of HK receptor activation using  - galactosidase as a marker gene.  AHK vs ZmHK. AHK3 and AHK4 available in pPIN-III vector. ZmHK1 and ZmHK3a were cloned and tested in the same expression vector. +/- IPTG induction.

8. Receptor testing - Arabidopsis  different ligand preferences. 3MeOBAP not active in AHK4.  no activity of N9 derivatives.

9. Receptor testing - maize  maize vs Arabidopsis. Maize HK receptors are more sensitive to aromatic cytokinins than Arabidopsis HK.  no activity of N9 derivatives.

10. Biological activity of N9 protected cytokinins - SUMMARY  active in in vitro cytokinin assays. Tobacco callus assay and Amaranthus assay.  show comparable properties as unprotected parent cytokinins on plant development. Positive cytokinin-like effects on plant development + reduced negative effects on root morphology.  no activity in HK receptor assays. Is there a mechanism accountable for the apparent contradiction in bioactivity vs ligand properties?

11. Cytokinin content in maize roots after treatment with 3MeOBAP and 3MeOBA9THPP T = 1 h T = 3 h T = 24 h T = 72 h * * * * ?

12. Cytokinin content in maize leaves after treatment with 3MeOBAP and 3MeOBA9THPP T = 1 h T = 3 h T = 24 h T = 72 h * * * * ?

13. Feeding experiments with radioactively labelled 3MeOBA9THPP … RT (min) Standard rootshootrootshoot 3-days application7-days application 2-3Adenine2.66%4.81%1.93%4.78% 4-6Adenosine, (OG)3OHBAP7.56%15.26%5.05%15.21% 8-93MeOBAPR5MP, 3OHBAP, (OG)3OHBAP1.80%3.19%0.68%1.47% 11-123MeOBAP9G, 3OHBAPR4.90%2.71%5.39%0.90% 18-193MeOBAP2.44%8.57%2.22%12.42% 20-213MeOBAPR1.75%0.73%0.43%0.56% 23-26Unknown8.34%40.18%8.02%27.46% 28-303MeOBA9THPP70.56%24.54%76.28%37.20%  The retention time (RT) of radioactivity detected after application of labeled 3MeOBA9THPP. Cytokinin standards eluted in corresponding RTs are listed together with level of detected radioactivity.

14. Expression profiles of genes involved in the cytokinin biosynthesis

15. Expression profiles of genes involved in the cytokinin biosynthesis

16. CONCLUSIONS  Some cytokinin derivatives at N9 can be metabolized in maize.  THP modification at N9 significantly enhances acropetal transport of a given cytokinin.  protection impedes the accumulation of non-active cytokinin glucoside forms in roots.  gradual release of the active base and its transport offer better distribution of the active cytokinin form.  amounts of naturally occurring isoprenoid cytokinins in different plant tissues are not strongly affected when plants are treated with protected cytokinin.

17. Thank you for your attention!