plant hormone

1. Cytokinins
Regulators of Cell Division
Cytokinins are substituted adenine compounds
that promote cell divisions in specific plant
tissues.

2. Discovery

3. History
• 1892: Weibner thought that cell division is regulated by endospermic compounds.
• 1913:
• 1941: Discovery by Johannes van Overbeek – coconut milk could sustain cell division
and prolonged growth of stem explants (excised pieces of stem)
IAA could not produce this effect.
• 1950s: Folke Skoog and coworkers – identified a modified purine (nucleotide) and
called Kinetin (Raven Fig. 28-8)
• 1913: Gottlieb Haberlandt discovered that a compound found in phloem had the
ability to stimulate cell division (Haberlandt, 1913).
• 1941:, Johannes van Overbeek discovered that the milky endosperm from coconut
also had this ability. He also showed that various other plant species had compounds
which stimulated cell division (van Overbeek, 1941).
• 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).

4. Structures of Some Naturally
Occurring Cytokinins

5. All cytokinins have basic Adenine ring structure

6. Structures of some naturally occurring cytokinins. Most plants have trans-zeatin as
the principal free cytokinin, but dihydrozeatin and isopentenyl adenine (i6Ade) are
also native plant cytokinins. Free cytokinins also include the ribosides and ribotides of
zeatin, dihydrozeatin, and isopentenyladenosine, although these may be active as
cytokinins by conversion to the respective bases. Free cytokinins from bacteria
include 2-methylthios-ribosylzeatin, as well as cis- or trans-zeatin, and their ribosides
and ribotides.

8. Structures of Some Synthetic
Cytokinins

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

10. Occurrence

11. Occurrence
• Cytokinins have been found in almost all higher plants as well as
mosses, fungi, bacteria, and also in tRNA of many prokaryotes
and eukaryotes.
• 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.
• Cytokinins have been found in ferns like Equisetum (horsetail)
and Dryopteris
• The balance of cytokinins and auxins acting together causes
development of organs like shoots and roots

13. Cytokinins Are Also Present in Some tRNAs in Animal and Plant
Cells
• Most organisms contain a variety of modified bases in their tRNA.
The normal RNA bases are incorporated into the newly synthesized
tRNA chain, and then specific enzyme modify a small subset of the
bases. As with the free cytokinins, Δ2-isopentenyl groups are
transferred to the adenine molecules. The enzyme catalyzing this
reaction is called tRNA-IPT, which is weakly similar in its sequence
to the plant cytokinin biosynthetic enzyme. The similarity was used
to help identify the cytokinin biosynthetic enzyme from plants. The
tRNA-IPT must be able to recognize a specific base sequence in the
tRNA and transfer the isopentenyl group to the adenosine nearest
the 3' end of the anticodon. It does not utilize free AMP as a
substrate.
• The possibility that the free cytokinins are derived from tRNA has
been explored extensively. Although the tRNA-bound cytokinins can
act as hormonal signals for plant cells if the tRNA is degraded and
fed back to the cells, it is unlikely that any significant amount of the
free hormonal cytokinin in plants is derived from the turnover of
tRNA.

14. Some tRNAs contain a cytokinin. The cytokinin in the examples shown is
[9R] iP. Although cytokinins are present in tRNA, tRNA is probably not
the source of free cytokinins in plants. (After Hall et al. 1967.)

15. Biosynthesis

16. Site of Synthesis
• Occurs in root tips and developing seeds
Transport
• Via xylem from root to shoot
Active & Passive Transport System

17. • 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
• Chen and Meltiz (1979) – Tobacco tissues
contain an enzyme that forms isopentenyl
adenosine-5-p from AMP and isopentenyl
pyrophosphate
• Isopentenyl AMP is converted into isopentenyl
adenine
Biosynthesis

19. 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.

20. Bioassay

21. • 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

22. PERCEPTION AND SIGNAL
TRANSDUCTION

23. • A, Evidence that CRE1 is a cytokinin receptor. The left-most
pathway depicts the osmosensing pathway in wild-type yeast: the
His kinase SLN1, which suppresses the activity of SSK2 (a mitogen-
activated protein kinase kinase kinase) activity via a phosphorelay
consisting of YPD1 (an Hpt) and SSK1 (a response regulator). A
deletion mutant of SLN1 is lethal due to overactivation of SSK2.
CRE1 can suppress the growth defect in an SLN1 deletion only in
the presence of cytokinins (two right pathways). B, Model of
cytokinin signaling in Arabidopsis. Cytokinin binds to the N-terminal
domain of CRE1 (and likely other similar sensor kinase) and
activates its His kinase activity. CRE1 phosphorylate the AHPs
(Arabidopsis Histidine phosphotransfer proteins), which in turn
transfer the phosphate to the receiver domain of ARR1 (Arabidopsis
response regulator) (and presumably to other type-B ARRs), thus
activating their output (transcriptional activator) domain. Type-A
ARRs (and possibly other primary target genes) are transcriptionally
induced by the activated type-B ARRs. The type-A ARRs also
interact with the AHPs, and are also likely phosphorylated. The
activated type-A ARRs, perhaps in parallel and/or in combination
with the activated type-B proteins, interact with various effectors to
alter cellular function, including the a feedback inhibition of their own
expression. The curved arrows indicate phosphotransfer. CK,
Cytokinins. See text for additional details.

25. Summary of perception and signal
transduction
• Binding of cytokinin to CRE1 or other Related His Kinases
• Initiation of phosphorelay
• 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

26. Mechanism of Action

27. • Amount in plants of 0.01 to 1.0 mM
• tRNA cytokinins are considered primitive as they
occur in bacteria and have similar function as in
higher plants. They are not translocated, so not
true hormones.
• 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.

28. • 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 s3
cyclin, a G1-type cyclin.
• Evidence is also that cytokinins increase the
ability of CDKs of the G2 stage by activating
phosphatase which removes one P from inactive
site to form the CDKs-cyclin complex
• 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

29. Physiological Roles

30. 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

31. The illustration shows the effect of cytokinin and auxin
concentration on tissue culture experiments (Mauseth, 1991)

32. Cytokinin Can Promote Light-Mediated Development
• The det mutants of Arabidopsis develop many of the characteristics
of light-grown seedlings when they are germinated in the dark. The
det mutants have short, unhooked hypocotyls and expanded
cotyledons, and the apical meristem initiates leaves that
subsequently expand without light. Although the det mutants do not
develop full photosynthetic competence in the dark (recall that the
enzymatic conversion of protochlorophyllide a to chlorophyllide a is
directly activated by light), there is partial development of the
chloroplasts, including the expression of genes encoding many
photosynthetic enzymes that are not present in the chloroplasts of
dark-grown wild-type seedlings.
• When wild-type Arabidopsis seedlings are germinated in darkness in
the presence of cytokinin, they develop many of the characteristics
of the det mutants: Hypocotyls shorten, cotyledons expand, the
apical meristem initiates leaves, and there is partial development of
the chloroplasts, including the synthesis of some photosynthetic
enzymes. This effect is proportional to the cytokinin dosage given to
the seedlings (Chory et al. 1994). Cytokinin is said to produce a
phenocopy of the det mutations.

33. • The ability of exogenous cytokinin to cause de-etiolation of dark-
grown seedlings is mimicked by certain mutations that lead to
cytokinin overproduction. Dark-grown Arabidopsis seedlings
carrying the amp1 mutation have zeatin levels that are
approximately 2.5 times higher than those of the wild type and
develop the characteristics of det mutant seedlings (Chory et al.
1994). Although the det mutants do not have elevated levels of
cytokinins, they may have altered sensitivity to cytokinins, since
Arabidopsis tissues from det1 plants, when cultured in vitro, do not
require cytokinin for growth and become green.
• Although the functions of the genes altered by the det mutants are
not completely understood, the det2 mutation is in a gene required
for the synthesis of brassinosteroids. Brassinosteroids and
cytokinins may have antagonistic effects in regulating light-induced
development. These results suggest that cytokinins may be part of
the light signal transduction pathway that is responsible for the
initiation of normal vegetative development and photosynthetic
competence. However, the role for cytokinins in the developmental
pathway initiated by light is not clearly established.

34. The effects of cytokinin on the development of wild-type Arabidopsis seedlings grown in darkness. (A–C)
The appearance of the seedlings after 1, 2, and 3 weeks, respectively, of growth in the dark with increasing
concentrations of cytokinin. The control (no cytokinin) is on the left in each case. The next five seedlings
were treated with 3, 15, 30, 60, and 75 µM of cytokinin, respectively. As the cytokinin concentration was
increased, the inhibition of hypocotyl elongation became more pronounced, while the cotyledons expanded
somewhat and leaves were initiated from the shoot apical meristem. At the higher cytokinin concentrations
the seedlings were phenocopies of det mutants. Cytokinin treatment also resulted in thylakoid formation in
the plastids of dark-grown seedlings (E) as compared to the development of plastids as etioplasts in the
untreated, dark-grown wild-type control (D). (From Chory et al. 1994, courtesy of J. Chory, © American
Society of Plant Physiologists, reprinted with permission.)

35. 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).
The source of the increase in cytokinin level in the bud has not yet
been determined. Much of the cytokinin of the plant is synthesized in
the root and transported to the shoot. Studies with the 14C-labeled
cytokinin benzyladenine (BA), have shown that when the labeled
compound is applied to roots, more [14C]BA is transported to the
shoot apex than to the axillary bud. Decapitation increases the
accumulation of [14C]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.