Cytokinins Biosynthesis, Signaling Pathways, Functions and Cross talk with Auxins

ARSHAD MAHMOOD KHAN

arshadbotanist@gmail.com

PhD Scholar (13-arid-3581)

BOTANY DEPARTMENT

UAAR RAWALPINDI

LECTURE OUTLINE

CYTOKININS

● Cytokinins ○ Discovery ○ Chemical Structure ○ Biosynthesis ○ Transport ○ Signaling ○ Functions

DISCOVERYCYTOKININS

• Haberlandt (1913) – compound in phloem stimulates cell division

• Van Overbeek (1941) – coconut milk (endosperm) also has the ability to stimulate cell division

• Jablonski & Skoog (1954) – compounds in vascular tissues promote cell division

• Miller (1955) – first cytokinin isolated from herring (Fish) sperm, named kinetin

●Miller (1955) – first cytokinin isolated from herring sperm, named kinetin ●Miller (1961) – first naturally occurring cytokinin found in plants (Zea mays

L.), later called Zeatin ●Lathum & Guillfoyle (1974)Lathum & Guillfoyle (1974)

ZeatinZeatin

DISCOVERYCYTOKININS

CHEMICAL STRUCTURECYTOKININS

●Adenine structure (amino purine ring) ●N6 Side chain

○Degree of unsaturation ○Number of carbons in side chain

CHEMICAL STRUCTURECYTOKININS

Free base forms are most hormonally

active (Yamada et al. 2001)

May have sugars attached to make

molecule more inactive and useful for

transport

Ribosides Ribotides

Glycosides

BIOSYNTHESISCYTOKININS

Biosynthesis started by the IPT (isopentenyl tranferase) enzyme

Major pathway Figure on slide no. 9

Other pathway

tRNA biosynthesis Figure on slide no. 10

BIOSYNTHESIS

Plant physiology, 4th E

di. Fig. 21.6, 2006 Sinauer A

ssociate. Inc

BIOSYNTHESIStRNA breakdown not the major pathway for

cytokinin synthesis

Plant physiology, 4th E

di. Fig. 21.7, 2006 Sinauer A

ssociate. Inc

BIOSYNTHESISCYTOKININS

●Generated mostly in the root apical meristems but also found in:

○Root cap cells ○Ovules

○Phloem cells ○Leaf axils

○Tips of young inflorescences ○Fruit

○Seeds

TRANSPORTCYTOKININS

●Cytokinins move up the plant through the xylem

●By contrast, auxin moves from top down.

Some signal in the shoot can also induce cytokinin transport from the root (Beveridge 2000).

SIGNALING PATHWAY

Cytokinins perception and pathwayCytokinin Signaling Pathway in Arabidopsis consists of four steps:

1. Initiation of a Phosphorelay Cascade by distinct Plasma membrane His (Histidine) Protein Kinases upon Cytokinin perception

2. Convergence of the signals initiated at His Protein Kinases, on AHP proteins, that serve as Phospho-relay carriers between the Cytokinin receptors and the downstream nuclear responses

3. Activation of B-type ARR proteins by nuclear AHP

4. Translocation and transcriptional activation of A-type ARRs

Mechanism:• First of all Cytokinins binds with the CHASE (Cyclases/Histidine

kinases–Associated Sensory Extracellular) domain of receptors e.g. (CRE1, AHK2, AHK3) present in PM

• The binding of Cytokinin activates the Transmembrane Domain, which Autophosphorylates on a His domain.

• The phosphate then transferred to Receiver Domain and an AHP protein, which translocates to the nucleus, where it activates ARRs.

• ARR form a large gene family composed of 22 genes that includes two major classes (Type-A and Type-B)

• Type-A ARRs (ARR3,4, 5, 6, 7,8,9,15,16,17,18,19) • Type-B ARRs (ARR1, ARR2, and ARR10,11,12,13,14)

AHP activates Type-B ARRs increases the transcription of the

Type-A ARRs, which feed back to inhibit their own transcription.

This transcription and translation of specific ARRs produces

specific cytokinins responses in plants

Cytokinins perception and pathway

●Cytokinin changes the structure of the His Kinase domain

○Phosphate moves from the kinase domain to the receiver domain ○Phosphate moves onto AHP ○AHP moves from cytoplasm into nucleus ○Phosphate on AHP moves onto receiver domain of the response regulator ○Change in structure of the receiver domain affects the output domain ○Output domain then signals transcription to cytokinin responses

SIGNALING SUMMARY CYTOKININS

FUNCTIONSCYTOKININS

●FUNCTIONS ○Cell division ○Root and shoot meristems ○Cell differentiation ○Leaf senescence ○Inflorescence growth ○Nutrient mobilization ○Cotyledon expansion ○Apical dominance ○ Seedling morphology

● Auxin and cytokinins influence the activity of:

○Cyclin-dependant protein kinases (CDKs) and cyclins

Both are proteins that regulate transitions between G1 to S and G2 to mitosis stages in the cell cycle

○Auxin stimulates the production of CDKs and cyclins

Cytokinins activate CDKs and cyclins through phosphrorylation and allow transition between stages

CELL DIVISIONCYTOKININ FUNCTION

Fig. 15-7, p. 241

cyclin

cyclinsynthesized

aminoacids

cyclin

(active)phosphorylation

ATP?GTP?

(inactive)

cyclindegraded

dephosphorylationtriggers mitosis

or DNA synthesis

P

cyclin

C-PK

CDK CDK CDK

26S Proteosome

● Optimal levels of cytokinins are needed for normal cell division

○Root: cytokinin overabundance inhibits cell division ○Shoot: cytokinins promote cell division

● Cytokinin oxidase dictates meristemic cytokinin concentrations

○Mutants can either overproduce or underproduce this enzyme

ROOT AND SHOOT MERISTEMSCYTOKININ FUNCTION

ROOT AND SHOOT MERISTEMS

CYTOKININ FUNCTION

Fig. 15-10, p. 244

CELL DIFFERENTIATIONCYTOKININ FUNCTION

Bacteria: Agrobacterium tumefaciens

●Auxin:cytokinin affects cell differentiation in callus tissue ●More auxin leads to roots development●More cytokinin leads to shoots development

●Skoog and Miller (1965)

CELL DIFFERENTIATIONCYTOKININ FUNCTION

LEAF SENESCENCECYTOKININ FUNCTION

Cytokinin delays leaf senescence Delay the degradation of chloroplasts May increase the growing season for agricultural purposes.

LEAF SENESCENCECYTOKININ FUNCTION

• Delayed leaf senescence, help plants to recover from environmental stresses like

•Drought (Rivero et al. 2007)

• Flooding (Zhang et al. 2000) • Hyunh et al. 2005

Control Sprayed

●Cytokinins induce division in inflorescence tips

○Leads to more flowering which yields more fruit

●“Cytokinin Oxidase Regulates Rice Grain Production” (Ashikari et al. 2005)

○Plants in this study have lower levels of cytokinin oxidase. The plants then produce more fruit.

INFLORESCENCE GROWTHCYTOKININ FUNCTION

●Promotes expansion in cotyledons ●Extends the cell wall ●Differs from auxin expansion

○Unlike auxin, no proton extrusion through the cell wall happens

COTYLEDON EXPANSIONCYTOKININ FUNCTION

●Antagonistic hormone interaction between cytokinin and auxin

○Cytokinin stimulates growth in auxiliary buds

Inhibits shoot elongation ○Auxin restrains growth in auxiliary buds

Causes shoot to lengthen. Mutants that overproduce cytokinins in lateral meristems are bushy.

APICAL DOMINANCECYTOKININ FUNCTION

●Etiolated leaves treated with cytokinins produce more active/productive chloroplasts upon illumination ●Dark-germinated seedlings treated with cytokinins

○Shortened hypocotyls ○Expanded cotyledons

Partial development of etioplasts into chloroplasts

SEEDLING MORPHOLOGYCYTOKININ FUNCTION

SEEDLING MORPHOLOGYCYTOKININ FUNCTION

1 (control) 2 (most concentrated)

3 4 (most dilute)

Average Hypocotyle

(mm)

8.8 2.9 5.4 8.1

Standard

Deviation

(mm)

1.398 0.567 0.699 0.875

Molarity

(mol/L)

none 4.651x10-4 4.651x10-5 4.651x 10-6

●Ashikari, Motoyuki. “Cytokinin Oxidase Regulates Rice Grain Production.” Science. 23 June 2005. 1 May. 2009 <http://www.sciencemag.org/cgi/content/abstract/309/5735/741> ●Davies, Peter J. ed. Plant Hormones. Boston: Kluwer Academic Publishers, 2004 ●Ma, Qing-Hu. “Genetic Engineering of Cytokinins and Their Application to Agriculture.” Critical Reviews in Biotechnology. 28.3 (2008) InformaWorld. University of California Santa Cruz Lib., Santa Cruz, CA, 1 May. 2009 <http://www.informaworld.com/smpp/sectioncontent=a904079709&full text=713240928> ●Mok, David W. S. , Machteld C. Mok, eds. Cytokinins Chemistry, Activity, and Function. Boca Raton:CRC Press Inc, 1994

REFERENCESCYTOKININS

Thanks for listening!

Thanks for your attention!Questions/Comments