Chapter 21

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 …..

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Arabidopsis.info

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

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First cytokinin discovered -- kinetin. Not reported in plants Reported in human urine.

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N

N

N

N

H

CH2NH

O

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 …..

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Zeatin isomerase trans form generally predominates

Zea and Oryza – cis form

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Derivatives of adenosine monophosphate (AMP). R determines type

isoprenoid cytokinin. aromatic cytokinin – least common

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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

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Thidiazuron

Occur both bound and free Some plant pathogens secrete free

cytokinins! Agrobacterium Fungi Insects Nematodes

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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

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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

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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!

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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

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Removes inhibitory phosphate

Promotes accumulation of G1 cyclins (CycD)

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.

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Sugarcane callus

High IAA and High Cytokinin

Cytokinins

High IAA and Low Cytokinin

Development of ShootsCytokinins

High IAA and no Cytokinin

Shoots ElongateCytokinins

High IAA and no Cytokinin

Development of RootsCytokinins

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?

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Witches broom in Pinus strobus

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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

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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.

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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 …..

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Delayed senescence appears to deal with distribution of nutrients Exogenous

application Spots stay green Nutrients (and AA)

migrate to spot Cytokinins stimulate

metabolism?28

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

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Cytokinin maintains meristem by controlling cell division

Deficient mutants – lonely guy (LOG)

Gene activates cytokinins

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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

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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

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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.

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Totipotency

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

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

nodules

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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

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