Cytokinins: Regulators of Cell Division Chapter 21

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

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

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

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 …..

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

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

Cytokinin Structure

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

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

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

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

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!

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 -- 12-24 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

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

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.

Organo-genesis

High IAA and High Cytokinin Cytokinins Sugarcane callus High IAA and High Cytokinin

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

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

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

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?

Biological effects of cytokinins Witches broom in Pinus strobus

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

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.

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 …..

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?

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

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

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

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

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.

Cytokinins Totipotency

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

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

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