Cytokinins

• Cytokinins are regulators of cell division but not elongation as auxins and GA do. It can cause
elongation of some excised cells (not in vivo).

• The 1st cytokinin to be discovered was not in a plant, it was from a herring fish autoclaved
(sterilized) sperm DNA. When this sperm DNA ages, it produces 6-furfuryladenine (kinetin)
→Cytokinin is made up of adenine (an aminopurine present in DNA) and a furfuryl group.
6-furfuryl adenine was applied to tobacco culture cells and a callus was produced in the
culture medium (occurred without having auxins in the medium). This compound was also
called kinetin.

• The 1st cytokinin from plant came from the milky endosperm of Zea mays (corn). The seeds
of this endosperm are soft (milky and rich with cytokinins). This cytokinin was called Zeatin,
and it was the first naturally occurring cytokinin to be derived from plants.

• Zeatin, and the other naturally occurring cytokinins, are active in the free base form, but can
be present in nucleoside or nucleotide form (mono-di-triphosphate).

• 2nd naturally occurring cytokinin discovered: Isopentenyl adenine (IPA). Unlike zeatin, it
doesn’t have an OH group.
• 3rd naturally occurring cytokinin discovered: dihydrozeatin. It is fully hydrogenated → no
double bond.
• These three are the naturally occurring cytokinins.
• Zeatin can be present as trans (more active form) or cis zeatin (found at higher
concentrations). The enzyme zeatin isomerase converts one form to another.
• The isoprene part is very important. It is transferred by isopentenyl transferase.

• Synthetic cytokinins:
- 6-furfuryl adenine
- Benzyl-adenine
• All cytokinins contain an adenine group (they are derived from adenine). They promote the
formation of buds. For a callus to grow, we need IAA. The differentiation of the callus is based
on the ration of IAA to cytokinins.
Cytokinins delay senescence in leaves. They also promote cell division and bud formation and
cell elongation in excised dicot cotyledons.
Some cytokinins are found close to tRNA. When they hydrolyze tRNA, some resulting N-bases
are cytokinins. They are close to anticodons.
• Cytokinins are not only produced by plants. They may also be found in pathogenic bacteria,
fungus, microrrhizea (bacteria and plant), animals, giant cells of nematodes, and galls of
insects (cytokinins are required for galls, which are organs that play a role in feeding).

• Cytokinins were mainly understood after the crown gall disease. It is caused by the bacterium
agrobacterium tumefaciens, which gets inside the plant through a wound in stem. Suppose
a plant cell is infected by the bacteria. Note that there is a T plasmid in the bacterial cell so
the bacteria can survive special environmental conditions. In the plastid of this bacterium,
there is a T-DNA gene. Part of the plasmid (T-DNA) is incorporated in the plant cell gene
sequence. So the plant cell is now a transformed plant cell. Now this DNA contains genes that
are necessary for the synthesis of auxins and one enzyme that is necessary for the synthesis
of cytokinins (Isopentenyltransferase IPT). IPT transfers the IP group (5 carbons) from IPP to
AMP, ADP, or ATP. It will lead to the production of two enzymes needed to synthesize auxins.
Also, there are genes in this piece of DNA that are needed for the synthesis of nitrogenous
compounds called opines (octopines and nopalines), and these are used as nitrogen source
for the bacterial cells.
So T-DNA contains genes for: 1 enzyme for cytokinin synthesis, 2 enzymes for IAA synthesis
from tryptophan, and opines.
Now with the production of auxins and cytokinins, the cells here will divide, and a tumor like
growth will grow, resulting in the formation of the crown gall à crown gall disease. So, that
disease led to the discovery of isopentenyltransferase, which is an essential enzyme in
cytokinins synthesis.
Why is this piece of bacterial DNA expressed in the transformed plant cell? Why is it not
expressed in the bacteria? The promoters of T-DNA are present in the plant genome à plant
eukaryotic promoters.

• Biosynthetic Pathway:
- To synthesize zeatin, we need adenine and a donor of the isopentenyl group:
The enzyme IPT (isopentenyl transferase) transfers this group from a donor and adds it to
adenine. This donor is AMP in bacteria, and ADP or ATP in plant cells. The donor of the
isopentenyl group is ∆2-IPP (like in gibberellins).

- For IPA (isopentenyl adenine) synthesis: we need a hydrolytic enzyme (phosphatase) to remove
the phosphate groups and another hydrolytic enzyme to remove the ribose. In plants, we remove
3 phosphates and ribose.

- We can synthesize zeatin from IPA by a hydroxylase enzyme and a donor of the hydroxyl group.

- If we want to synthesize dihydrozeatin, we need a reductase enzyme to act on zeatin and

saturate the double bond.

• Synthesis occurs in the root tips (mainly in the meristematic cells), and then they are
transported to other parts of the plant. The transport occurs through the xylem. It could take
place also in young embryos (zeatin à present in the milky seed of Zea mays). It is also
reported to take place in young developing leaves and young fruits (all of these have
meristems).

• Inactivation:
- Cytokinin can be inactivated by conjugation. This way it’ll be stored for later use. The can form
conjugates with small molecules such as glucose. Both O and N glycosides are storage forms that
can be activated.

- Cytokinin can be also inactivated through oxidation by cytokinin oxidase. It is an irreversible

reaction. This enzyme can cleave the isopentenyl group of zeatin (both cis and trans) as well as
their N-glycoside derivatives but not their O glycoside derivatives.

Cytokinin --------> Adenine + 3-methyl-2-butenal

• Physiological Effects of Cytokinins:

1) Regulate cell division:
It does so by regulating the cell cycle. Cyclin dependent kinases (CDKs) (enzymes that
regulate cell cycle) production is controlled by cytokinins. However, note that both auxins
and cytokinins are needed for cell division.
Cell Division Cycle 2 (CDC 2) (a major CDK): expression of this gene is controlled by IAA
(auxin). CDC 2 is produced in the phosphorylated form (inactive). Then, Cytokinins will
 induce the phosphatase gene → phosphatase will remove the inhibitory phosphate that
is attached to CDC2 enzyme, making it active.

2) Plant tissue culture:

Plant tissue culture is important for synthesis of GMO (genetically modified organism).
Cytokinins and auxins are needed for plant tissue culture, in addition to inorganic and
organic compounds (sorbitol as a source of carbon, and glutamate as a source of
nitrogen).
Coconut milk is rich in cytokinins. It is usually added to the medium.
At the beginning, we form a mass of undifferentiated cells: callus. To control
differentiation of cells, we can control the ratio of cytokinins to auxins:
If the ratio of cytokinins to auxins is high → buds will differentiate to shoots
If the ratio of cytokinins to auxins is low → buds will differentiate to roots

3) Cell elongation or enlargement in excised cells (in vitro):

If we add cytokinins to excised cotyledons of radish, pumpkin, sugar beets, flax,
lettuce…they will elongate → increase in size not in number.
Cytokinins induce invertase enzyme → inverts sucrose to glucose and fructose → solute
concentration increases → water potential decreases → water enters the cells (water
uptake) → elongation occurs.

4) Seed germination:
Example in lettuce seeds: two important factors should be present for the seed to
germinate: water and oxygen. In presence of these two factors, cytokinins can promote
germination if applied to the seed by promoting the expression of certain genes through
a cascade of events.
Also, red light can promote the buildup of endogenous cytokinins, this is done through
phytochrome pigment: PR → Pfr →→ cytokinins.

5) Lateral bud development:

Lateral bud development is inhibited by apical dominance, which is: the presence of IAA
in the tip will lead to high concentration of ABA in the buds, and ABA causes bud
dormancy.
Cytokinins promote lateral bud development, even if they were dormant, but for the
growth to continue, we should add auxins or GA (depending on the plant).

6) Retention of chlorophyll and delayed senescence in leaves:

 If we detach a leaf from the plant → breakdown of nutrients, nitrogenous compounds,
proteins, and this will be followed by breakdown of chlorophyll (chlorosis), the leaf will
no longer remain green in color and it will age and eventually die.
If you excise leaves and add cytokinins, this will call the chlorophyll to remain green and
aging will be delayed.
If you initiate the formation of roots at the petiole (base of the leaf) which can be done
by adding a certain concentration of cytokinins, then this will result in the delaying of
senescence. This is because most of the cytokinins are produced at the root tips, so
nutrients are mobilized, and these cytokinins will be transported to the leaf, which
remains green for more time.
There’s a disease that affects cereals: rust disease → the leaf will have brown spots like
rust (necrosis). This disease is caused by the fungus paxinogramas that belongs to
basediomycetus family. These necrotic spots are surrounded by green areas / green
islands that are very rich in starch. Why are they rich in starch? The fungus produces
cytokinins and cause starch to be transported to these areas so that the fungus can
survive. Recall the cytokinins are also produced by fungi, bacteria, insects, nematodes,
and others. This is proof that cytokinins can delay senescence.

7) Promoting chloroplast development and chlorophyll synthesis:

The synthesis of the chlorophyll requires light; light is necessary for reductase enzyme to
be expressed (this enzyme is necessary for chlorophyll synthesis in angiosperms). If you
grow a plant in the dark, this seedling will not develop a green color. It will remain yellow.
The seedling is said to be etiolated. This yellow color is due to the presence of
carotenoids. In these etiolated seedling, we have etioplasts instead of chloroplasts; these
are characterized by lacking chlorophyll and the presence of an interesting organelle
called the prolamellar body. The etioplast, upon exposure to light, will develop into a
chloroplast and the prolamellar body will develop into the thylakoid membrane which will
in turn form the grana.
Cytokinins cannot replace the role of light. But cytokinins will enhance the development
of the etioplasts into chloroplasts and increases the rate of chlorophyll synthesis. But both
should happen in the presence of light.