1. Cytokinins: Regulators of Cell Division
Chapter 21

2. Cell Division & Plant Development
Differentiated cells can resume division
Wounding induces division at the wound site
Generally self-limiting
Agrobacterium tumefaciens infection
Diffusible factors control cell division
Division stops because signal stops?
Plant tissues and organs can be cultured
Roots could be cultured; stems were recalcitrant
Crown gall tissue, OTOH …..
Arabidopsis.info

3. Cytokinins and Tissue Culture
Roots – grow fine with no hormones
Shoots – no growth even with meristems
Unless adventitious roots present
 difference between regulation in shoot and root
 root derived factors
regulate shoot
Crown Gall tissue

4. Cytokinins: Background
First cytokinin discovered -- kinetin.
Not reported in plants
Reported in human urine. N H
CH2 O

5. Discovery of Cytokinins
Coconut endosperm (coconut milk) – supported continued division of mature, differentiated cells
1940s-50s – Adenine had some effect
Aged herring sperm …..
Kinetin
Corn endosperm (1973) – zeatin
Notice the double bond …..

6. Zeatin Zeatin isomerase trans form generally predominates
Zea and Oryza – cis form

7. Cytokinins: Background
Derivatives of adenosine monophosphate (AMP).
R determines type
isoprenoid cytokinin.
aromatic cytokinin – least common

8. Cytokinin Structure

9. What is a cytokinin? Biological activities similar to trans-zeatin
Inducing cell division in the presence of auxin
Promoting bud or root formation in appropriate ratios to auxin
Delaying leaf senescence
Promoting expansion of dicot cotyledons
Almost all are aminopurine deriviatives
Thidiazuron

10. Cytokinins Occur both bound and free
Some plant pathogens secrete free cytokinins!
Agrobacterium
Fungi
Insects
Nematodes

11. Cytokinins Found in actively dividing tissues
E.g., seeds, fruits, leaves, root tips,
Found in bleeding sap (think wound healing!)
Occur in algae, mosses, horsetails, ferns, and conifers

12. Sites of cytokinin synthesis
Primary site of synthesis -- root tips.
High concentrations -- immature seeds and developing fruits
synthesis *OR* transport?
Evidence indicates that locally produced cytokinins required to release buds from dormancy

13. Biological effects of cytokinins
Cytokinins only stimulate cell division in the presence of auxin!
Tobacco tissue culture – callus only
In the absence of meristem or cambium – cell division happened!

14. Biological effects of cytokinins
Specific role -- regulate the progression of the cell cycle.
In absence of either auxin or cytokinin -- G1 or G2
Supply missing hormone hours – division begins
Activate cyclin-dependent kinases (CDK).
Activation of CDK – allows transition from G2 to mitosis.
Promotion of accumulation of cyclins allow transition from G1 to S

15. Biological effects of cytokinins
Removes inhibitory phosphate
Promotes accumulation of G1 cyclins (CycD)

16. Biological effects of cytokinins
Auxin/cytokinin interaction regulates formation of tissues in cell culture.
Both hormones in similar concentrations -- maintain cells as undifferentiated callus.
High auxin -- only plant roots form.
High cytokinin -- only shoots form.
Proper ratios – callus can produce an intact plant.

17. Organo-genesis

19. High IAA and High Cytokinin
Cytokinins
Sugarcane callus
High IAA and High Cytokinin

20. High IAA and Low Cytokinin
Cytokinins
Development of Shoots
High IAA and Low Cytokinin

21. High IAA and no Cytokinin
Cytokinins
Shoots Elongate
High IAA and no Cytokinin

22. High IAA and no Cytokinin
Cytokinins
Development of Roots
High IAA and no Cytokinin

23. Biological effects of cytokinins
Cytokinins and apical dominance.
Application of cytokinin to meristem or to the axillary bud will release the bud!
Plants overproducing auxin – increased apical dominance
Plants overproducing cytokinin – decreased dominance
Application of cytokinin to plants overproducing auxin releases buds as well
Such results illustrate that the ratio of auxin to cytokinin
“witch’s broom”
Extreme lateral bud release
overproduction of cytokinin?

24. Biological effects of cytokinins
Witches broom in Pinus strobus

25. Biological effects of cytokinins
Crown gall tumors -- Agrobacterium tumifaciens.
Excised tumors can exist with no added hormones
Contain tumor-inducing (Ti) plasmid
3 classes of proteins
Auxin and cytokinen production
Opines – nutrients for the bacterium
Plasmid inserted into nuclear DNA upon infection

26. Biological effects of cytokinins
Cytokinins are involved in the formation of crown gall tumors in plants.
Transferred DNA (T-DNA) from the plasmid is incorporated into the host plant genome.
The T-DNA contains three genes.
Two of the genes are necessary for the synthesis of auxins and cytokinins.
The third gene causes the plant to produce opines, which are a nutrient amino acid for the bacteria.
The natural ability of A. tumifaciens to genetically reprogram plants can been adapted to facilitate the transfer of other genes into plants.

27. Biological effects of cytokinins
Cytokinins -- inhibitors of senescence.
Exogenous cytokinins delay senescence.
On detached leaves – no senescence
Leaves treated with auxin (for adventious root formation) – no senescence
Leaves spotted with cytokinin – spots stay green
20 week tobacco plants …..

28. Biological effects of cytokinins
Delayed senescence appears to deal with distribution of nutrients
Exogenous application
Spots stay green
Nutrients (and AA) migrate to spot
Cytokinins stimulate metabolism?

29. Biological effects of cytokinins
Meristem – delicate balance between dividing cells and differentiating cells
Cytokinins play a part
Reducing in vivo concentrations
increased degradation or loss-of-function receptors
Retarded or halted shoot development
Dwarf, late flowering plants
Reduced size of the shoot meristem
Slowed formation of leaf primordia
A reduced number of leaf cells

30. Biological effects of cytokinins
Cytokinin maintains meristem by controlling cell division
Deficient mutants – lonely guy (LOG)
Gene activates cytokinins

31. Evo-Devo
Homeotic genes – genes that control placement and spatial organization of body parts by controlling the developmental fate of groups of cells
Contain 180 nucleotide sequence called a homeobox
Homeobox – specifies homeodomain in the protein
Homeodomain – part of the protein that binds to the DNA when the protein functions as transcriptional regulator
Can bind to any DNA segment
Other domains determine which genes the protein regulates
Plant homeobox genes include some MADS-box genes and KNOX genes

32. KNOX genes
KNOX genes -- regulate biosynthesis of cytokinins and gibberellins to specify meristems
KNOX genes -- normally expressed in SAM but not leaf primordia or developing leaves.
A dominant, gain-of-function mutation
expression of KNOX genes in developing leaves, causing “knots” of cells or ectopic shoots to form.
Accumulation of KNOX proteins – division only, no differentiation
Loss-of-function mutants
A defect in STM (ShootMeristemless) prevents formation of apical meristem

33. Biological effects of cytokinins
KNOX proteins -- transcription factors that regulate transition from indeterminate growth to differentiation.
KNOX proteins :
Up-regulate IPT genes for cytokinin synthesis
Suppress the GA20-oxidase gene in gibberellin synthesis
KNOX proteins -- maintain the high cytokinin/low gibberellin ratio needed for formation and maintenance of shoot meristems.

34. Cytokinins
Totipotency

35. Functions of Cytokinins
Summary from Taiz & Zeiger
Promote shoot growth by increasing cell proliferation in the SAM
Inhibit root growth by promoting the exit of cells from the RAM
Regulate components of the Cell Cycle
Modify apical dominance and promote lateral bud growth
Delay leaf senescence

36. Functions of Cytokins Summary from Taiz & Zeiger
Promote movement of nutrients
Affect light signaling
Regulate vascular development
Involved in the formation of nitrogen-fixing nodules

37. Cytokinins
Chemical Nature: N6-adenine derivatives, phenyl urea compounds. Zeatin is the most common cytokinin
Sites of Biosynthesis: primarily in root tips
Transport: transported in the xylem from roots to shoots
Effects: promotion of cell division; promotion of shoot formation in tissue culture; delay of leaf senescence; application of cytokinin can cause release of lateral buds from apical dominance and can increase root development in arid conditions