Biol 352 Lecture 6 Cytokinins: Regulators of Cell Division January 24, 2007

1. Biol 352 Lecture 6 Cytokinins: Regulators of Cell Division January 24, 2007

2. Plant Hormones • What we expect you to know: • The basic terminology, concepts, principles, and processes • The unique features of a hormone • Biosynthesis: The critical points in a biosynthesis pathway • Metabolism: The critical enzymes or steps • Transport: The uniqueness • Functions: The basic functions, experimental approaches • Signal transduction: critical components, major processes • What we do not expect from you: • Draw the chemical structure • Draw the whole biosynthesis pathway • Memorize the long name of a chemical • Memorize all the intermediate molecules in a biosynthesis pathway

3. Review:Gibberellins • GA structure feature • Biosynthesis: 3 steps, 3 cellular compartments, 3 key enzymes • Function: stem elongation, seed germination • Signal transduction: transcriptional repressor, protein degradation

4. Lecture Outline: • Definition of cytokinin • Cytokinin biosynthesis • Functions of cytokinin • Cytokinin signal transduction • Summary

5. Learning Objectives: • Define the key enzyme in cytokinin biosynthesis • Define the key enzyme in cytokinin degradation • Use experimental approaches to demonstrate the functions of cytokinin • Be familiar with the principle of phosphorelay two-component signalling system • Reading: • 4th Ed, Plant Physiology, Taiz & Zeiger, Chapter 21: p543-567 • (3rd Ed, Plant Physiology, Taiz & Zeiger, Chapter 21: p493-515)

6. Identification of Cytokinins • Coconut milk, 1940s • The first cytokinin to be discovered was Kinetin (as a breakdown product of DNA). • 3) Kinetin is nota naturally occurring cytokinin. • 4) Zeatin is the most abundant natural cytokinin. 6 5 7 1 8 2 4 9 3 Kinetin (6-furfury-laminopurine)

7. 6 5 7 1 8 2 4 9 3 Adenine (alternate name: 6-aminopurine) (Chemical formula: C5H5N5) Kinetin (6-furfury-laminopurine)

8. Structure of cytokinins: • All the naturally occurring cytokinins are aminopurine derivatives. • Nearly all compounds active as cytokinins are N6-substituted aminopurine. Adenine (alternate name: 6-aminopurine)

9. (Z) (iP) (DZ)

10. Cytokinins: compounds that have biological activities similar to those of trans-zeatin, including: • Induce cell division in callus cells in the presence of an auxin • Promote bud or root formation from callus cultures when in the appropriate molar ratios to auxin • Delay senescence of leaves • Promote expansion of dicot cotyledons

11. Forms of Cytokinins:Cytokinins can be present in the plant as: • Riboside—a ribose sugar is attached to the N-9 of the purine ring • Ribotide—the ribose sugar moiety contains a phosphate group • Glycoside—a sugar molecular is attached to the 3, 7, 9 nitrogen of the purine ring, or to the oxygen of the zeatin or dihydrozeatin side chain 6 5 7 1 8 2 4 9 3 Kinetin (6-furfury-laminopurine) Ribose

12. iPA ZR ZMP

13. Cytokinin Biosynthesis • Major sites of synthesis: root apical meristem, young developing leaves, young fruits • 1st step: transfer of the isopentenyl group of dimethylallyl diphosphate (DMAPP) to an adenosine moiety by isopentenyl transferase (IPT) • The biosynthesis involves interconversion among bases, ribosides, and ribotides, side chain modification, and conjugation.

14. Cytokinin Biosynthesis • AMP/ADP/ATP plus DMAPP act as precursors for synthesis of iP ribotide (iPMP/iPDP/iPTP) • Bacterial ipt gene and plant IPTgenes are involved in cytokinin synthesis IPT: isopentenyl transferase

15. Cytokinin Biosynthesis • Interconversion • Side chain modification • Conjugation

16. Cytokinin Oxidase

17. Left: WT • Middle:AtCKX1 overexpressing plant • Right:AtCKX2 overexpressing plant …Cytokinin oxidase genes reduce [active Cytokinins] and retard plant growth

18. Functions of Cytokinins: • Promote cell division • Together with auxin regulate morphogenesis in cultured tissues • Delay leaf senescence • Promote cell expansion in leaves and cotyledons • Promote chloroplast development • Modify apical dominance

19. 1.Cytokinins Regulate Cell Division WT Overexpression of AtCKX1

20. 1.Cytokinins Regulate Cell Division • Left: wild-type • Right: Overexpression of cytokinin oxidase

21. 2. Auxin/Cytokinin Ratio Regulates Morphogenesis in Cultured Tissues High Auxin/Cytokinin: Root Low Auxin/Cytokinin: Shoot Intermediate Auxin/Cytokinin: Callus [Kinetin] [IAA]

22. 3.Cytokinins Delay Leaf Senescence

23. Journal of Anti-Aging Medicine Mar 2002, Vol. 5, No. 1: 113-116 Suresh I.S. Rattan N6-Furfuryladenine (Kinetin) as a Potential Anti-Aging Molecule

24. 4. Cytokinins Promote Cell Expansion Light Dark control + zeatin • Cytokinin-induced wall loosening is not accompanied by proton extrusion • Neither auxin nor GA promotes cell expansion in cotyledons

25. Cytokinin Signal Transduction AHP: histidine phosphotransfer protein ARR: Arabidopsis response regulator

26. Cytokinin phosphorelay two-component signalling system CRE1: cytokinin receptor AHP: Arabidopsis histidine phosphotransfer protein ARR: Arabidopsis response regulator Type-B ARR: receiver domain and transcription factor domain; transcription factor Type-A ARR: receiver domain only; primary response genes

28. 0 2 5 10 15 25 30 40 60 120 180 min following cytokinin treatment ARR4 ARR5 Type-A ARR genes in response to cytokinin ARR6 Northern blot ARR7 ARR16 Tubulin Expression of ARR5 Promoter:GUS reporter In situ mRNA hybridization

29. Summary: • Cytokinins are N6-substituted aminopurines that initiate cell proliferation together with auxin. • Zeatin is the most abundant natural cytokinin. • 1st committed step in cytokinin biosynthesis is catalyzed by isopentenyl transferase (IPT). • Cytokinin oxidase degrades cytokinin. • Cytokinin regulates cell division, morphogenesis, cell expansion, and leaf senescence. • Cytokinin signalling use a phosphorelay two-component signalling system.