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

DISCOVERY CYTOKININS 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

DISCOVERY CYTOKININS 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) Zeatin

CHEMICAL STRUCTURE Adenine structure (amino purine ring) N6 Side chain CYTOKININS Adenine structure (amino purine ring) N6 Side chain Degree of unsaturation Number of carbons in side chain

CHEMICAL STRUCTURE Free base forms are most hormonally CYTOKININS 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

BIOSYNTHESIS CYTOKININS Biosynthesis started by the IPT (isopentenyl tranferase) enzyme Major pathway Figure on slide no. 9 Other pathway tRNA biosynthesis Figure on slide no. 10

Plant physiology, 4th Edi. Fig. 21.6, 2006 Sinauer Associate. Inc BIOSYNTHESIS

BIOSYNTHESIS tRNA breakdown not the major pathway for cytokinin synthesis Plant physiology, 4th Edi. Fig. 21.7, 2006 Sinauer Associate. Inc

BIOSYNTHESIS Generated mostly in the root apical meristems CYTOKININS 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

TRANSPORT 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 \figures\ch21\pp21270.jpg

Cytokinins perception and pathway Cytokinin Signaling Pathway in Arabidopsis consists of four steps: Initiation of a Phosphorelay Cascade by distinct Plasma membrane His (Histidine) Protein Kinases upon Cytokinin perception 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 Activation of B-type ARR proteins by nuclear AHP 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.

Cytokinins perception and pathway 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

Cytokinin changes the structure of the His Kinase domain SIGNALING SUMMARY CYTOKININS 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

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

CELL DIVISION CYTOKININ FUNCTION 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

26S Proteosome cyclin synthesized amino acids cyclin degraded cyclin triggers mitosis or DNA synthesis dephosphorylation CDK CDK CDK P (inactive) (active) phosphorylation Figure 15.7: Diagram showing how the cdc2 protein kinase (C-PK) and cyclin trigger cell cycle progression in cycling cells. Starting in the top left, cyclin has joined to C-PK in a phosphorylated form to make a new, but inactive, protein. An enzyme removes the phosphate (P) to activate this protein. This form of the protein acts as the trigger to switch on S phase and M phase. It is recycled by separating C-PK, which is phosphorylated again and recycled. The cyclin is broken down into amino acid subunits and must be resynthesized. ATP, Adenosine triphosphate; GTP, guanosine triphosphate. C-PK ATP? GTP? Fig. 15-7, p. 241

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ROOT AND SHOOT MERISTEMS CYTOKININ FUNCTION 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 MERISTEMS CYTOKININ FUNCTION

Figure 15. 10: The interplay of auxin and cytokinin Figure 15.10: The interplay of auxin and cytokinin. Differences in the amounts of auxin and cytokinin can change the development of tobacco (Nicotiana tabacum) tissues in culture. Fig. 15-10, p. 244

CELL DIFFERENTIATION Bacteria: Agrobacterium tumefaciens CYTOKININ FUNCTION Bacteria: Agrobacterium tumefaciens

Auxin:cytokinin affects cell differentiation in callus tissue CYTOKININ FUNCTION Auxin:cytokinin affects cell differentiation in callus tissue More auxin leads to roots development More cytokinin leads to shoots development Skoog and Miller (1965)

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

LEAF SENESCENCE CYTOKININ FUNCTION Control Sprayed 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

Cytokinins induce division in inflorescence tips INFLORESCENCE GROWTH CYTOKININ FUNCTION 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.

COTYLEDON EXPANSION CYTOKININ FUNCTION Promotes expansion in cotyledons Extends the cell wall Differs from auxin expansion Unlike auxin, no proton extrusion through the cell wall happens

APICAL DOMINANCE CYTOKININ 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.

SEEDLING MORPHOLOGY CYTOKININ 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 MORPHOLOGY CYTOKININ FUNCTION 1 (control) 2 (most concentrated) 3 4 (most dilute) Average Hypocotyle (mm) 8.8 2.9 5.4 8.1 Standard Deviation 1.398 0.567 0.699 0.875 Molarity (mol/L) none 4.651x10-4 4.651x10-5 4.651x 10-6

REFERENCES CYTOKININS 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 Thanks for listening!

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