EFB530 Plant Physiology

Cytokinins and cell division

Discovery

Mature plant cells usually do not divide

• most cell division occurs within the meristems / cambial layers
• under some circumstances, cells will be reinitiated to divide
• early experiments (1913) indicated that this reinitiation was caused by a diffusible factor (a hormone) produced by particular tissues

Plant tissue culture is a powerful experimental tool, allowing manipulation and proliferation of plant cells

• in the 1930s, it was discovered that tomato roots could be cultured in a defined medium indefinitely, continuing to produce differentiated root tissue
• this was not the case with shoots - they would not grow (until they developed roots)

Many additives were tried in the effort to get shoot tissue to grow in culture

• one additive that worked was coconut milk (discovered in the 40's)
• Carlos Miller, a student of Folke Skoog discovered in 1954 that autoclaved (heat + pressure) DNA supported the growth of stem tissue in culture
• the active ingredient was purified from the autoclaved DNA = kinetin (kinetin is not naturally produced in plants)
• growth stimulation by kinetin requires auxin also
• the natural cytokinin in coconut milk (also discovered in maize endosperm by Carlos Miller in early 70's) is zeatin

Cytokinins (like auxins) are defined more by their biological activity (inducing cell division in tissue culture) rather than by structure

• nearly all have a purine with a side group

Zeatin is the abundant cytokinin found in most plants

• synthesized from AMP and an attached isoprene derivative

Synthetic cytokinins include kinetin and benzylaminopurine (BAP)

Cytokinins are primarily synthesized in the root apical meristem, then are transported through the plant in the xylem sap

Role of cytokinins

1) cytokinins (together with auxin) probably regulate the cell cycle

• cell cycle = G1 -> S (DNA synthesis) -> G2 -> M (mitosis) (-> back to G1)
• the cell cycle may pause at two times (check points/restriction points), during G1 or during G2 (before mitosis)
• observation of cell metabolism in tissue culture indicates that auxin may promote advancement to S = promoting DNA replication
• cytokinin appears to promote advancement through M = cytokinesis (tissue culture cells deprived of cytokinin stop at G2 checkpoint)
• auxin up-regulates the expression of a cyclin-dependent kinase (CDK) gene
• cytokinin up-regulates the expression of a cyclin gene

2) cytokinins (together with auxin) may regulate tissue morphogenesis, since the ratio of auxin : cytokinin in tissue culture medium determines root or shoot production

• high auxin : cytokinin = root production
• intermediate auxin : cytokinin = callus growth
• low auxin : cytokinin (high cytokinin) = shoot production

3) cytokinins delay senescence and promote nutrient uptake

• application of cytokinin to a leaf on an aging plant can allow that leaf to stay green, while the others yellow and die
• cytokinin application to lateral buds can promote their growth
• some pathogens produce cytokinins that attract nutrients to that tissue or cause extensive growth of laterals buds (leads to witch's broom)[Rhodococcus fascians]

4) cytokinins promote chloroplast maturation and cell enlargement in leafy cotyledons

• cytokinin application promotes development of chloroplasts from etioplasts
• sections of leafy cotyledons enlarge when treated with cytokinin (but not acid growth)

Cytokinin receptors

The cytokinin receptors have been identified and they are like the two-component signalling system common in bacteria

• characteristic in this system is a receptor with a histidine kinase domain
• when activated by binding the hormone, the kinase transfers a phosphate to an aspartate, then to a phosphotransfer protein
• the phosphotransfer protein then phosphorylates the response protein, which becomes active as a transcription factor or may affect some other cellular response to cytokinin
• there are multiple cytokinin receptors and many different response proteins, implying redundancy of function and/or specialization of response functions

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