Plant Biochemistry
Unit IV
Lecture I
Cytokinins:
Regulators of Cell Division
THE CYTOKININS WERE DISCOVERED in the search for factors that stimulate plant cells to divide (i.e.,
undergo cytokinesis). Since their discovery, cytokinins have been shown to have effects on many other
physiological and developmental processes, including leaf senescence, nutrient mobilization, apical dominance, the
formation and activity of shoot apical meristems, floral development, the breaking of bud dormancy, and seed
germination. Cytokinins also appear to mediate many aspects of light-regulated development, including chloroplast
differentiation,the development of autotrophic metabolism, and leaf and cotyledon expansion.
Although cytokinins regulate many cellular processes, the control of cell division is central in plant growth and
development and is considered diagnostic for this class of plant growth regulators. For these reasons we will preface
our discussion of cytokinin function with a brief consideration of the roles of cell division in normal
development,wounding, gall formation, and tissue culture.
Later in the chapter we will examine the regulation of plant cell proliferation by cytokinins. Then we will turn to
cytokinin functions not directly related to cell division: chloroplast differentiation, the prevention of leaf senescence,
and nutrient mobilization. Finally, we will consider the molecular mechanisms underlying cytokinin perception and
signaling.
THE DISCOVERY, IDENTIFICATION, AND PROPERTIES OF CYTOKININS
A great many substances were tested in an effort to initiate and sustain the proliferation of normal stem tissues in
culture. Materials ranging from yeast extract to tomato juice were found to have a positive effect, at least with some
tissues.
However, culture growth was stimulated most dramatically when the liquid endosperm of coconut, also known as
coconut milk, was added to the culture medium. Philip White’s nutrient medium, supplemented with an auxin and
10 to 20% coconut milk, will support the continued cell division of mature, differentiated cells from a wide variety
of tissues and species, leading to the formation of callus tissue (Caplin and Steward 1948). This finding indicated
that coconut milk contains a substance or substances that stimulate mature cells to enter and remain in the cell
division cycle.
Eventually coconut milk was shown to contain the cytokinin zeatin, but this finding was not obtained until several
years after the discovery of the cytokinins (Letham 1974). The first cytokinin to be discovered was the synthetic
analog kinetin.
, Kinetin Was Discovered as a Breakdown Product of DNA
In the 1940s and 1950s, Folke Skoog and coworkers at the University of Wisconsin tested many substances for
their ability to initiate and sustain the proliferation of cultured tobacco pith tissue. They had observed that the
nucleic acid base adenine had a slight promotive effect, so they tested the possibility that nucleic acids would
stimulate division in this tissue. Surprisingly, autoclaved herring sperm DNA had a powerful cell division–
promoting effect. After much work, a small molecule was identified from the autoclaved DNA and named
kinetin. It was shown to be an adenine (or aminopurine) derivative, 6-furfurylaminopurine (Miller et al. 1955):
In the presence of an auxin, kinetin would stimulate tobacco pith parenchyma tissue to proliferate in culture.
No kinetin-induced cell division occurs without auxin in the culture medium. (For more details, see Web
Topic 21.1.) Kinetin is not a naturally occurring plant growth regulator, and it does not occur as a base in the
DNA of any species. It is a by-product of the heat-induced degradation of DNA, in which the deoxyribose
sugar of adenosine is converted to a furfuryl ring and shifted from the 9 position to the 6 position on the
adenine ring.
The discovery of kinetin was important because it demonstrated that cell division could be induced by a simple
chemical substance. Of greater importance, the discovery of kinetin suggested that naturally occurring
molecules with structures similar to that of kinetin regulate cell division activity within the plant. This
hypothesis proved to be correct.
Zeatin Is the Most Abundant Natural Cytokinin
Several years after the discovery of kinetin, extracts of the immature endosperm of corn (Zea mays) were found
to contain a substance that has the same biological effect as kinetin. This substance stimulates mature plant
cells to divide when added to a culture medium along with an auxin. Letham (1973) isolated the molecule
responsible for this activity and identified it as trans-6-(4-hydroxy-3- methylbut-2-enylamino)purine, which he
called zeatin:
The molecular structure of zeatin is similar to that of kinetin. Both molecules are adenine or aminopurine
derivatives. Although they have different side chains, in both cases the side chain is attached to the 6 nitrogen
of the aminopurine. Because the side chain of zeatin has a double bond, it can exist in either the cis or the trans
configuration.
In higher plants, zeatin occurs in both the cis and the trans configurations, and these forms can be
interconverted by an enzyme known as zeatin isomerase. Although the trans form of zeatin is much more
active in biological assays, the cis form may also play important roles, as suggested by the fact that it has been
found in high levels in a number of plant species and particular tissues. Agene encoding a glucosyl transferase
enzyme specific to cis-zeatin has recently been cloned, which further supports a biological role for this isoform
of zeatin.
Since its discovery in immature maize endosperm, zeatin has been found in many plants and in some bacteria.
It is the most prevalent cytokinin in higher plants, but other substituted aminopurines that are active as
cytokinins have been isolated from many plant and bac-terial species.
Unit IV
Lecture I
Cytokinins:
Regulators of Cell Division
THE CYTOKININS WERE DISCOVERED in the search for factors that stimulate plant cells to divide (i.e.,
undergo cytokinesis). Since their discovery, cytokinins have been shown to have effects on many other
physiological and developmental processes, including leaf senescence, nutrient mobilization, apical dominance, the
formation and activity of shoot apical meristems, floral development, the breaking of bud dormancy, and seed
germination. Cytokinins also appear to mediate many aspects of light-regulated development, including chloroplast
differentiation,the development of autotrophic metabolism, and leaf and cotyledon expansion.
Although cytokinins regulate many cellular processes, the control of cell division is central in plant growth and
development and is considered diagnostic for this class of plant growth regulators. For these reasons we will preface
our discussion of cytokinin function with a brief consideration of the roles of cell division in normal
development,wounding, gall formation, and tissue culture.
Later in the chapter we will examine the regulation of plant cell proliferation by cytokinins. Then we will turn to
cytokinin functions not directly related to cell division: chloroplast differentiation, the prevention of leaf senescence,
and nutrient mobilization. Finally, we will consider the molecular mechanisms underlying cytokinin perception and
signaling.
THE DISCOVERY, IDENTIFICATION, AND PROPERTIES OF CYTOKININS
A great many substances were tested in an effort to initiate and sustain the proliferation of normal stem tissues in
culture. Materials ranging from yeast extract to tomato juice were found to have a positive effect, at least with some
tissues.
However, culture growth was stimulated most dramatically when the liquid endosperm of coconut, also known as
coconut milk, was added to the culture medium. Philip White’s nutrient medium, supplemented with an auxin and
10 to 20% coconut milk, will support the continued cell division of mature, differentiated cells from a wide variety
of tissues and species, leading to the formation of callus tissue (Caplin and Steward 1948). This finding indicated
that coconut milk contains a substance or substances that stimulate mature cells to enter and remain in the cell
division cycle.
Eventually coconut milk was shown to contain the cytokinin zeatin, but this finding was not obtained until several
years after the discovery of the cytokinins (Letham 1974). The first cytokinin to be discovered was the synthetic
analog kinetin.
, Kinetin Was Discovered as a Breakdown Product of DNA
In the 1940s and 1950s, Folke Skoog and coworkers at the University of Wisconsin tested many substances for
their ability to initiate and sustain the proliferation of cultured tobacco pith tissue. They had observed that the
nucleic acid base adenine had a slight promotive effect, so they tested the possibility that nucleic acids would
stimulate division in this tissue. Surprisingly, autoclaved herring sperm DNA had a powerful cell division–
promoting effect. After much work, a small molecule was identified from the autoclaved DNA and named
kinetin. It was shown to be an adenine (or aminopurine) derivative, 6-furfurylaminopurine (Miller et al. 1955):
In the presence of an auxin, kinetin would stimulate tobacco pith parenchyma tissue to proliferate in culture.
No kinetin-induced cell division occurs without auxin in the culture medium. (For more details, see Web
Topic 21.1.) Kinetin is not a naturally occurring plant growth regulator, and it does not occur as a base in the
DNA of any species. It is a by-product of the heat-induced degradation of DNA, in which the deoxyribose
sugar of adenosine is converted to a furfuryl ring and shifted from the 9 position to the 6 position on the
adenine ring.
The discovery of kinetin was important because it demonstrated that cell division could be induced by a simple
chemical substance. Of greater importance, the discovery of kinetin suggested that naturally occurring
molecules with structures similar to that of kinetin regulate cell division activity within the plant. This
hypothesis proved to be correct.
Zeatin Is the Most Abundant Natural Cytokinin
Several years after the discovery of kinetin, extracts of the immature endosperm of corn (Zea mays) were found
to contain a substance that has the same biological effect as kinetin. This substance stimulates mature plant
cells to divide when added to a culture medium along with an auxin. Letham (1973) isolated the molecule
responsible for this activity and identified it as trans-6-(4-hydroxy-3- methylbut-2-enylamino)purine, which he
called zeatin:
The molecular structure of zeatin is similar to that of kinetin. Both molecules are adenine or aminopurine
derivatives. Although they have different side chains, in both cases the side chain is attached to the 6 nitrogen
of the aminopurine. Because the side chain of zeatin has a double bond, it can exist in either the cis or the trans
configuration.
In higher plants, zeatin occurs in both the cis and the trans configurations, and these forms can be
interconverted by an enzyme known as zeatin isomerase. Although the trans form of zeatin is much more
active in biological assays, the cis form may also play important roles, as suggested by the fact that it has been
found in high levels in a number of plant species and particular tissues. Agene encoding a glucosyl transferase
enzyme specific to cis-zeatin has recently been cloned, which further supports a biological role for this isoform
of zeatin.
Since its discovery in immature maize endosperm, zeatin has been found in many plants and in some bacteria.
It is the most prevalent cytokinin in higher plants, but other substituted aminopurines that are active as
cytokinins have been isolated from many plant and bac-terial species.
