2. Plant Physiology II BS Botany 7 th semester Habib-ur-Rehman Athar Institute of Pure & Applied Biology Bahauddin Zakariya University, Multan Cytokinins Regulators of Cell Division Cytokinins

3. Cytokinins Cytokinins Cytokinins are substituted adenine compounds that promote cell divisions in specific plant tissues.

4. History 1892: Weibner thought that cell division is regulated by endospermic compounds. 1941: Discovery by Johannes van Overbeek – coconut milk could sustain cell division and prolonged growth of stem explants (excised pieces of stem) He also showed that various other plant species had compounds which stimulated cell division (van Overbeek, 1941). IAA could not produce this effect. 1950s: Folke Skoog and coworkers – identified a modified purine (nucleotide) and called Kinetin In 1954:, Jablonski and Skoog extended the work of Haberlandt showing that vascular tissues contained compounds which promote cell division (Jablonski and Skoog, 1954). 1955: The first cytokinin was isolated from herring sperm in 1955 by Miller and his associates (Miller et al., 1955). This compound was named kinetin because of its ability to promote cytokinesis. Hall and deRopp reported that kinetin could be formed from DNA degradation products in 1955 (Hall and deRopp, 1955). 1961: Miller isolated the first naturally occurring cytokinin from corn (Miller, 1961). It was later called zeatin. Almost simultaneous with Miller, Letham published a report on zeatin as a factor inducing cell division and later described its chemical properties (Letham, 1963). It is Miller and Letham that are credited with the simultaneous discovery of zeatin. Since that time, many more naturally occurring cytokinins have been isolated and the compound is ubiquitous to all plant species in one form or another (Arteca, 1996; Salisbury and Ross, 1992).

5. All cytokinins have basic Adenine ring structure

6. There are synthetic cytokinins derived from diphenylurea (DPU) that are structurally unrelated to the adeninetype cytokinins.

7. Occurrence Cytokinins have been found in almost all higher plants as well as mosses, fungi, bacteria, Cytokinins are found in actively growing tissues where cell division takes place (root tip, shoot tip, expanding leaf, developing endosperm – e.g. Liquid endosperm of coconut, immature maize endosperm. Today there are more than 200 natural and synthetic cytokinins combined. However it is not known whether they are synthesized in these tissues or transported to these tissues from other sites of synthesis. Root tips are the probable sites of cytokinin synthesis. The balance of cytokinins and auxins acting together causes development of organs like shoots and roots

9. Site of Synthesis Occurs in root tips and developing seeds Transport Via xylem from root to shoot

10. Cytokinins occur in free form or in tRNA The major site of biosynthesis of free cytokinins is root tip and distribute via xylem – also produced in developing buds, developing seeds. tRNA-cyto are formed in every living cell in cytoplasm, chloroplast and mitochondria Biosynthesis

12. Proposed biosynthetic and metabolic pathway for cytokinins. Left, The proposed biosynthesis of zeatin tri-/diphosphate in Arabidopsis. Both ADP and ATP are likely substrates for the plant IPT enzyme, and these and their di- and triphosphate derivatives are indicted together (e.g. ATP/ADP). The biosynthesis of cytokinins in bacteria (e.g. A. tumefaciens) is compared next to it. Right, Several possible modifications and the degradation of zeatin. The diagram only depicts reactions that are described in the text; cytokinin metabolism is more complex than the pathways shown (see Mok and Mok, 2001). See text for more details.

13. There are many bioassays available for the estimation of cytokinins activity some of these are: –Tobacco pith callus –Radish cotyledon expansion They are directly related to the role of cytokinins in cell division Specific metabolic bioassay is –β-cyanin synthesis in Amaranthus seedlings –Chlorophyll retention in oat leaves

14. Summary of perception and signal transduction Binding of cytokinin to CRE1 or other Related His Kinases Initiation of phosphorylation Phosphorylation and activation of the type-B ARRs (Arabidopsis response regulators) Transcription of Type-A genes which in case over-expression negatively feedback the signaling pathway Type-A and Type-B ARRs interact with various molecules (effectors) inside the cell and determine the kind of biochemical reactions in response to cytokinin

16. Cytokinins are 0.01 to 1.0  in plants Most important function of cytokinins is cytokinesis. Application of cytokinins promote cell division by increasing the change of cell from G2 to mitosis This is done by enhancing protein synthesis, since specific enzymes are required for mitosis. Cytokinins effect on translation but not on transcription. Ribosomes frequently grouped together to form long polysomes but yet no information about specific enzymes. Evidence showed that cytokinins are involved in the regulation of the cell cycle. They control the activity of cyclin-dependent kinases (CDKs) Cytokinis promote cell division by stimulating the expression of the genes that gives rise to  3 cyclin, a G1-type cyclin. Recently it has been found that cytokinins stimulate the expression of the CYCD3 gene which encodes a D-type cyclin which plays a role in cell cycle in G1 stage.  D-type cyclins play a major role in regulation of cell proliferation Physiological Roles/ Mode of Action

17. Cytokinin Functions Stimulates cell division Promote cell expansion: only in dicot seedlings but in stem and roots they inhibit cell inhibit cell expansion probably due to production of ethylene in stem and roots Promote Chloroplast maturation: Promotes the conversion of etioplasts into chloroplasts via stimulation of chlorophyll synthesis - Etiolated leaves treated with cytokinins develop chloroplast Stimulates morphogenesis (shoot initiation/bud formation) in tissue culture. Stimulates the growth of lateral buds-release of apical dominance. Stimulates leaf expansion resulting from cell enlargement. May enhance stomatal opening in some species. Involved in releasing seed dormancy Delay of senescence Induction of enzymes and gene expression – stimulate RNA and protein synthesis – post-transcriptional regulation Promote nutrient metabolism in some species

18. Role of Cytokinins in Apical Dominance Measurements of cytokinin levels in axillary buds of Douglas fir (Pseudotsuga menziesii) show a very good correlation between endogenous cytokinin levels and bud growth (Pilate et al. 1989). Much of the cytokinin of the plant is synthesized in the root and transported to the shoot. Studies with the 14 C-labeled cytokinin benzyladenine (BA), have shown that when the labeled compound is applied to roots, more [ 14 C]BA is transported to the shoot apex than to the axillary bud. –Decapitation increases the accumulation of [ 14 C]BA by the axillary bud, and application of auxin to the apical stump reduces this accumulation. –Thus auxin makes the shoot apex a sink for cytokinin from the root, and this may be one of the factors involved in apical dominance.