Biosynthesis, translocation, physiological role in cell level, basic functions and mechanism of action of cytokinins

1. Biosynthesis, translocation, physiological role in
cell level, basic functions and mechanism of action
of cytokinins.
BY
A P GOKUL
1 st Ph. D Horticulture

2. Biosynthesis
Translocation
 simple compounds are modified, converted into other compounds,
or joined to form macromolecules
 a biological mechanism involving the transfer of water and other
soluble nutrients from one part of the plant to another through the
xylem and phloem

3. CYTOKININ
Role of Hormone Cell division (increase number of cells)
Site of Production Root Tips
Effect of Hormone
Mitosis of new cells;
Stimulates seed germination
new shoot growth

4. Cytokinins – Occurrence & Distribution
 Kinetin - first cytokinin discovered and named after its cytokinesis.
 Though it is a natural compound, It is not made in plants, and is therefore usually considered a
"synthetic" cytokinin.
 most common CK in plants today is called zeatin which was isolated from corn (Zea mays).
 CKs have been found in almost all higher plants as well as mosses, fungi, bacteria, and also in
tRNA of many prokaryotes and eukaryotes.
 Today there are more than 200 natural and synthetic cytokinins combined.
 Concentrations are highest in meristematic regions and areas of continuous growth potential
such as roots, young leaves, developing fruits, and seeds.

5. SITE OF
CYTOKININ
SYNTHESIS
 Primary synthesis site – root tips
 Higher concentrations – immature seeds and developing fruits.
 Naturally occuring cytokinin : coconut milk, tomato juice.
flowers and fruits of pear and
plum
cambium tissues of eucalyptus,
nicotina.
immature fruits of Zea Mays.

7. Two kinds
Adenine cytokinins
• Examples: Kinetin, Zeatin, IPA, DZ, BA (6- Benzylaminopurine).
The DNA base adenine is a structural analogue of cytokinins but have low cytokinin bioactivity.
Phenylurea cytokinins
• Example: N, N'- diphenylurea, Thidiazuron
Although their chemical compositions differ, there is a structural correlation between adenine and
urea cytokinins, and both show similar biological activities.
CYTOKININS
Three common examples: Kinetin (6-furfurylaminopurine),
BA (6-benzylaminopurine),
BPA (6-(benzylamino)-9-(2-tetrahydropyranyl)-9H-purine ).

8. zeatin
Isopentenyl
adenine
kinetin
Benzyl amino
purine
NATURAL OCCURING SYNTHETIC

9. Adenosine
precursor
Dimethylallyl
pyrophosphate (DMAPP)
Isopentyl adenosine-5-
diphosphate (iPDP)
ADP ATP
Isopentenyl
Transferase (ITP)
ADP
ATP
 Isopentenyl
Transferase (ITP)
Isopentyl adenosine-5-
triphosphate (iPDP)
Zeatin
Hydrolysis

10. Adenosine monophosphate
N6-delta-isopentenyl-adenine
Isopentenyl pyrophosphate
N6-delta-isopentenyl-adenosine
Zeatin
N6-delta-isopentenyl-adenosine-monophosphate
Isopentenyl transferase
Precursor
dephosphorylated
Hydroxylated
BIOSYNTHESIS OF ZEATIN AND OTHER NATURAL CYTOKININS

11. Cytokinins
• are not widely distributed in different plant parts
• Evidence show that cytokinins are synthesized in roots especially during seedling stage.
xylem
• Cytokinin have been found in xylem exudate and appear to be translocated through xylem.
Ribosides
signal
• Cytokinin are tranported in their bound form ribosides in plants. A signal from shoot regulates
the tranport.
• However, the identity of this signal remains to be determined

12.  When the shoot is cut from a rooted plant near the soil line, the xylem sap
may continue to flow from the cut stump for some time.
 This xylem exudate contains cytokinins. If the soil covering the roots is
kept moist, the flow of xylem exudate can continue for several days.
 Because the cytokinin content of the exudate does not diminish, the
cytokinins found in it are likely to be synthesized by the roots.
 In addition, environmental factors that interfere with root function, such as
water stress, reduce the cytokinin content of the xylem exudate.
(Itai and Vaadia 1971)

13.  To assess the role of cytokinin derived from the root, the tobacco root stock
engineered to overproduce cytokinin was grafted to a wild-type shoot.
 Surprisingly, no phenotypic consequences were observed in the shoot, even
though an increased concentration of cytokinin was measured in the
transpiration stream.
 Thus the excess cytokinin in the roots had no effect on the grafted shoot.
(Faiss et al. 1997)

14.  The cytokinins in the xylem exudate are mainly in the form of zeatin
ribosides.
 Once they reach the leaves, some of these nucleosides are converted to the
free-base form or to glucosides (Noodén and Letham 1993).
 Cytokinin glucosides may accumulate to high levels in seeds and in leaves,
and substantial amounts may be present even in senescing leaves.

15.  Evidence from grafting experiments with mutants suggests that the transport of
zeatin riboside from the root to the shoot is regulated by signals from the shoot.
 The rms4 mutant of pea (Pisum sativum L.) is characterized by a 40- fold
decrease in the concentration of zeatin riboside in the xylem sap of the roots.
 However, grafting a wild-type shoot onto an rms4 mutant root increased the
zeatin riboside levels in the xylem exudate to wild-type levels.
 Conversely, grafting an rms4 mutant shoot onto a wild-type root lowered the
concentration of zeatin riboside in the xylem exudate to mutant levels.
 These results suggest that a signal from the shoot can regulate cytokinin transport
from the root. The identity of this signal has not yet been determined.
(Beveridge et al. 1997)

16.  However, dihydrozeatin and its
conjugates are resistant to cleavage.
 Cytokinin oxidase irreversibly
inactivates cytokinins, and it could
be important in regulating or
limiting cytokinin effects.
 The activity of the enzyme is
induced by high cytokinin
concentrations, due at least in part to
an elevation of the RNA levels for a
subset of the genes.
 Free cytokinins are readily converted to their respective nucleoside and nucleotide forms. Such
interconversions likely involve enzymes common to purine metabolism.
 Many plant tissues contain the enzyme cytokinin oxidase, which cleaves the side chain from zeatin (both cis
and trans), zeatin riboside, iP, and their N-glucosides, but not their O-glucoside derivatives (Fig.).

17. THE BIOLOGICAL ROLES OF CYTOKININS

18.  Cytokinins are generally required for cell division of plant cells in vitro. Several lines of
evidence suggest that cytokinins also play key roles in the regulation of cell division in
vivo.
 Much of the cell division in an adult plant occurs in the meristems.
 Localized expression of the ipt gene of Agrobacterium in somatic sectors of tobacco
leaves causes the formation of ectopic (abnormally located) meristems, indicating that
elevated levels of cytokinin are sufficient to initiate cell divisions in these leaves
(Estruch et al. 1991).
 Elevation of endogenous cytokinin levels in transgenic Arabidopsis results in
overexpression of the KNOTTED homeobox transcription factor homologs KNAT1 and
STM—genes that are important in the regulation of meristem function (Rupp et al.
1999).
 Interestingly, overexpression of KNAT1 also appears to elevate cytokinin levels in
transgenic tobacco, suggesting an interdependent relationship between KNAT and the
level of cytokinins.

19.  Overexpression of several of the Arabidopsis cytokinin
oxidase genes in tobacco results in a reduction of
endogenous cytokinin levels and a consequent strong
retardation of shoot development due to a reduction in
the rate of cell proliferation in the shoot apical meristem
(Fig.) (Werner et al. 2001).

20.  Surprisingly, the same overexpression of cytokinin oxidase in
tobacco led to an enhancement of root growth (Fig.) primarily
by increasing the size of the root apical meristem (Fig.).
 Since the root is a major source of cytokinin, this result may
indicate that cytokinins play opposite roles in regulating cell
proliferation in root and shoot meristems.

21.  One of the primary determinants of plant form is the degree of apical dominance
(see Chapter 19). Plants with strong apical dominance, such as maize, have a
single growing axis with few lateral branches. In contrast, many lateral buds
initiate growth in shrubby plants
 Although apical dominance may be determined primarily by auxin, physiological
studies indicate that cytokinins play a role in initiating the growth of lateral buds.
For example, direct applications of cytokinins to the axillary buds of many
species stimulate cell division activity and growth of the buds.
 The phenotypes of cytokinin-overproducing mutants are consistent with this
result. Wild-type tobacco shows strong apical dominance during vegetative
development, and the lateral buds of cytokinin overproducers grow vigorously,
developing into shoots that compete with the main shoot. Consequently,
cytokinin-overproducing plants tend to be bushy.

22.  In the course of determining the structural requirements for cytokinin activity, investigators
found that some molecules act as cytokinin antagonist:
 3-Methyl-7-(3-methylbutylamino)pyrazolo[4,3-D]pyrimidine
 These molecules are able to block the action of cytokinins, and their effects may be
overcome by the addition of more cytokinin.
ANTAGONISTS/INHIBITORS

23. DEACTIVATION OF CYTOKININ
Conjugation (Reversible Irreversible)
By glucose or amino acid.
Oxidation (Irreversible)
By cytokinin oxidase
substrates of this enzyme
Trans and cis forms of zeatin riboside.
iP and their N- glucosides.

24. Cell
division
Cell
differentioation
Apical
dominance
Leaf
senescence
Nutrient
mobilization
Cotyledone
expansion
Inflorescence
growth
FUNCTIONS

27. 1. Promotion of cell division:
 The major physiological role of naturally occurring cytokinins is to
promote cell division.
 It is now well established that these are true cell division factors in a
number of lower and higher plants.
 In the presence of auxins, nearly all cytokinins stimulate cell division and
susbsequent callus growth in the parenchymatous cells of several plants.

28. 2. Cell enlargement:
 The promotion of cell enlargement by cytokinins is most clearly demonstrated
in the cotyledons of dicots with leafy cotyledons, such as mustard, cucumber,
and sunflower.
 The cotyledons of these species expand as a result of cell enlargement during
seedling growth.
 Cytokinin treatment promotes additional cell expansion, with no increase in the
dry weight of the treated cotyledons.
 Leafy cotyledons expand to a much greater extent when the seedlings are
grown in the light than in the dark, and cytokinins promote cotyledon growth
in both light- and dark-grown seedlings.

29. Conti…
 As with auxin induced growth,
cytokinin-stimulated expansion
of radish cotyledons is
associated with an increase in the
mechanical extensibility of the
cell walls.
 However, cytokinin-induced
wall loosening is not
accompanied by proton
extrusion. Neither auxin nor
gibberellin promotes cell
expansion in cotyledons

30. Control of morphogenesis
In plant tissue cultures, cytokinin is required for the growth of a callus
• callus + auxin + no cytokininlittle growth of callus
• callus + auxin + cytokinin callus grows well, undifferentiated
Ratio of cytokinin and auxin are important in determining the fate of the callus:
• callus + low [cytokinin/auxin]callus grows well, forms roots
• callus + high [cytokinin/auxin] callus grows well, forms meristem & shoots

31. Auxin and Cytokinin act
antagonistically, and the ratio
between the hormones is critical
for their effects.
Shoots
Increasing [Auxin]
Increasing
[Cytokinin]
Roots
Shoots
CK PROMOTES SHOOT GROWTH IN CULTURE

32. 4. Counteraction of apical dominance:
 External application of Cytokinins promotes the growth of lateral busd even if
the apical bud is intact.
 Sorokin and Thimann (1964) experimentally demonstrated that cytokinins
caused the formation of a vascular connection ( which was not allowed to be
formed by auxin released from the apical bud) which increases water and
solute supply for a renewed growth of the lateral buds.
 Thus, the cytokinins reverse the auxin induced inhibition of lateral buds and
counteract the apical dominance.

33. 5. Breaking the dormancy:
 Cytokinins can stimulate germination and break dormancy. It has the ability to change
the effects of other hormones without any marked effects on themselves.
 Gibberellins alone are not capable to overcome the thermo-dormancy or inhibitor
dormancy.
 Addition of cytokinin along with GA opposes the action of inhibitor and permits
germination.
 Thus cytokinin has been documented as a permissive agent in germination
antagonizing the inhibitor action (cytokinin-inhibitor antagonism).
 Cytokinins treatment, in dark overcomes the dormancy of certain light sensitive seeds
as littuce and tobacco.
 Dormant buds which remains inactive due to certain adverse factors, can be treated
with cytokinins to overcome the dormancy.

34. Case Study

35. Conti…

36. Delay senescence
• senescence is the programmed ageing process that occurs in plants
• loss of chlorophyll, RNA, protein and lipids.
• CK application to an intact leaf markedly reduces the extent and rate of chlorophyll
and protein degradation and leaf drop

37. IPT: isopentenyl transferase
Leaf senescence is retarded in a
transgenic tobacco plant containing ipt
gene from Agrobacterium tumefaciens
fused to senescence – induced promoter

38. • Leaf senescence is
retarded in
transgenic lettuce
plants expressing ipt

39. Control of leaf senescence by CKs
From Gan, S., and Amasino, R.M. (1995) Inhibition of leaf senescence by autoregulated production of cytokinin Science 270: 1986-1988. Reprinted
with permission from AAAS.
Plants that express IPT under the
control of a senescence-induced
promoter (SAG) have significantly
delayed leaf senescence.
SAG:IPT Control
SAG:IPT Control

40. The delay in senescence may be due to CK-induced invertase
CK induces invertase expression. Plants expressing invertase under the control of SAG12 show a
delay in senescence similar to that caused by SAG:IPT.
Lara, M.E.B., et al. (2004). Extracellular invertase is an essential component of cytokinin-mediated delay of
senescence. Plant Cell 16: 1276-1287.

41. Case Study

42. Conti….
 The final stage of leaf development is senescence, which can significantly affect the
survival, health, and productivity of plants during the growing season. Senescence is
characterized by color changes in both perennial and annual plants in the late summer
and throughout autumn. In this phase, the most perceptible phenotypic change that
embodies senescence is the appearance of variegated leaves, which develop due to the
disassembly of chloroplasts and the degradation of proteins, lipids, nucleic acids, and
pigments.
 The nutrients that are generated from the degradation of senescing leaves are
transported to developing seeds and fruits in annual plants or to new leaves or flowers
in perennial trees, resulting in the death of the senescing leaves.
 Leaf senescence is influenced by various endogenous signals (plant hormones and
age) and environmental signals (darkness, shading by other plants, UV-B or ozone
exposure, nutrient limitation, extreme temperatures, drought, high salinity, and
pathogen attacks)

43. Conti….
 Cytokinins are believed to serve as negative
regulators of leaf senescence in a variety of
monocotyledonous and dicotyledonous species.
 A reduction in cytokinin levels before the onset
of senescence is believed to be a key signal for
the initiation of senescence.
 The exogenous application of cytokinins or the
transgenic expression of cytokinin biosynthesis
genes prevents the degradation of chlorophyll,
photosynthetic proteins, and RNA, resulting in
delayed senescence

44. 7. Promotion of chloroplast development:
 Exogenously applied cytokinins promote chloroplast development in callus
tissues and excised cotyledons.
 Tobacco callus tissue in dark contains etioplasts devaid of chlorophyll with no
grana and lamellae.
 If it is treated with cytokinin in dark, promotes lamellar deveploment and if
light is also applied simultaneously ( with cytokinin) grana and chlorophyll
also appear.
 Thus, ctyokinin is essential for the transformation of etioplasts to chloroplasts.

45. Conti…

46. 8. Anthocyanin synthesis:
 Anthocynins are flavonoid pigments which are responsible for the red, pink,
purple and blue colours in plants.
 Cytokinin treatment increases anthocynin content in many culture cells and
tissues and in parts of intact plants.
 For example-in suspension cultured cells of carrot and sunflower, in the
seedlings of balsam and cauliflower and in the petals of rose, cytokinin
application increases anthocynin production.

47. Case Study

48. Conti…

49. Case Study

50. Conti…

51. Conti…

52. 9. Other roles/ effects:
 Some other roles or effects of cytokinins are- differentation of interfascicular
cambium and lignification, accumulatiom of solutes, stimulation of several
enzymes especially those concerned with photosynthesis, etc.

53. CK-mediated
processes
Reprinted from Werner, T., and Schmülling, T. (2009). Cytokinin action in plant
development. Current Opinion in Plant Biology 12: 527-538, with permission from Elsevier
copyright 2009.
There are many other processes
mediated by CK. Identifying the
specific genes that contribute to
each of these will help us to
understand the myriad roles that CK
plays in coordinating plant growth.