After the discovery of auxins and gibberellins and their influence on growth and development, efforts were diverted to find the factors that would stimulate plant cells to divide. These efforts led to the discovery of cytokinins, they were found to effect other physiological and developmental processes as well.
Folke Skoog tested many substances for their ability to stimulate cell divisions in tobacco pith tissue in a culture medium. He observed a slight promotive effect of the nucleic acid base adenine. So he tested the possibility that nucleic acids may stimulate division in this tissue. He used heat-tested herring (a fish) DNA and it stimulated cell division in tobacco pith tissue. A single chemical substance was isolated from the heated-treated DNA that in the presence of auxin identified as 6-furfurylaminopurine and named kinetin.
It was found that kinetin is active only in the presence of auxin and does not stimulate cell divisions in the absence of auxin. Kinetin is not an 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 the DNA in which the deoxyribose sugar of adenosine is converted to a furfuryl ring and shifted from the 9 to 6 position in the adenine ring. The discovery of kinetin is important because that cell division can be induced by a simple chemical substance.
Zeatin – The First Natural Cytokinin
Several years after discovery of kinetin, Carlos Miller in United States and D. S. Letham in Australia independently demonstrated that extracts of the immature endosperm of corn (Zea mays) contained a substance that had the same biological effect as kinetin. It stimulated mature plant cells to divide when added to a culture medium along with an auxin. Letham isolated the substance responsible for mitotic activity and identified it as 6-(4-hydroxy-3-methylbuta-trans-2-enylamino) purine, which he called zeatin. Its structure is similar to that of kinetin. Zeatin is the naturally occurring cytokinin discovered first and in most of the plants.
Since then, other cytokinins with aden8ne-like structures similar kinetin and zeatin have been identified in numerous parts of the seed plants. None of these cytokinins is present in DNA, some occur in tRNA and sometimes rRNA molecules of seed plants, yeast and bacteria.
A large number of chemical compounds have been synthesized and tested for cytokinin activity (synthetic cytokinin). Nearly all the compounds active as cytokinins are 6-substituted aminopurines. Benzylaminopurine (BAP), the most commonly used synthetic cytokinin in agriculture is an example of synthetic cytokinin.
Biosynthesis of Cytokinins
The side chains of cytokinins are constructed, at least in parts, from units of isoprene. The side chain of zeatin is similar to isoprene in structure. These side chains are synthesized from an isoprene derivatives. The precursor for the formation of these isoprene structures is mevalonic acid.
Mevalonic acid is converted into isopentenyl pyrophosphate (IPP) by a series of enzymatic reactions known as mevalonic acid pathway.
Enzymes cytokinins synthase transfer isopentenyl group of IPP to adenosine monophosphate to form ribosylzeatin-5-monophosphate (ZMP).
Ribosylzeatin-5-monophosphate (ZMP) is converted into zeatin (Z) through ribosylzeatin(ZR).
The synthesis of tRNA cytokinins takes place by an entirely different route. Free cytokinins are not used in the synthesis of cytokinin-active bases tRNA. Instead tRNA are made from four conventional nucleotides during the transcription of genes encoding their sequence. The first product is a tRNA precursor that lacks modified bases and is somewhat larger than the final product. This precursor is processed is yield to functional tRNA are modified to give the cytokinins.
Transport of Cytokinins
Cytokinins are syntheiszed in roots move through the xylem into the shoot. When the shoot is cut from a rooted plant near the soil line, the exudated xylem sap contains cytokinins. Also, environmental factors that interfere with root function, such as water stress, reduce the cytokinins contents of xylem exudate. Transport of zeatin and zeatin riboside certainly occurs in the xylem, but the sieve tubes contain cytokinins, especially glucoside . Further evidence for transport in phloem is indicated by experiments with detached dicot leaves. When a matu4re leaf is cut from the plant and kept moist, cytokinins move to the base of the petiole and accumulate there. However, if a radioactive cytokinins is added to the surface of a leaf, very little of that which is absorbed is transported out. This indicate that cyrokinins are readily distributed in the phloem.
Physiological Effects of Cytokinins
The cytokinins can regulate a variety of physiological, metabolic, biochemical and developmental processes when they are applied to higher plants. Some of these effects and responses are:
Regulation of Cell Cycles
The cell cycle is characterized by DNA replication and mitosis. Their is evidence that cyrokinins along with auxins and regulate the cell cycle. The auxin may regulate events leading to DNA synthesis whereas cytokinins regulate events leading to mitosis. For example, in cultured tobacco tissue, auxin initiate DNA synthesis but the cells divide only when they are provided with cytokinin. Similarly, if the tissue is treated with cytokinin, only cells with previously synthesized DNA will divide.
Regulation of Morphogenesis
Skoog found that callus formation is cultured tobacco pith cells, soybean and other dicot stems can be greatly promoted if cytokinins is provided along with auxin. He also found that if cytokinin-to-auxin ratio is maintained high, certain cells are produced in callus that divide into buds, stems and leaves. But if the cytokinin-to-auxin ratio is lowered, root formation is favoured.
The ability of the callus to form shoots and adventitious roots is called organogenesism. In some cases callus forms embryo that develop into a root and shoot, this is called embryogenesis.
Delay of Senescence
It has been observed that in dicot leaves appearance of adventitious roots at the base of the petiole delay senescence of the leaf blade. There are two main evidences that cytokinin is supplied by the roots through xylem to leaf that delay senescence. Firstly, if cytokinins are applied to leaves they replace the need for roots in delaying senescence and secondly when adventitious roots are formed; the cytokinin contents of leaf blade rises. Also if a single intact leaf is sprayed with cytokinin, it remains green whereas the other leaves of the same age becomes yellow and fall off.
Promotion of Lateral Bud Development in Dicots
Cytokiin can help in overcoming apical dominance as the hormone promotes the growth of lateral buds dominated by shoot apex above it. Kinetin, benzyladenine and zeatin have been used to promote growth of lateral bud.
A bacterium, Corynebacterium fascians, cause a disease known as fascination in chrysanthemum, garden peas and sweet peas, in which the normal rounded stems become flattened and numerous lateral buds develop into branches, forming a broom-like bundle of stems. It has been found that the bacterium contains a plasmid and it is able to synthesize several cytokinins, suggesting that cytokinins is responsible for lateral bud development.
Promotion of Chloroplast Maturation
The dark grown seedlings etiolated. In the absence of light etioplasts develop from proplastids of dark grown seedlings. These contains carotenoids and do not synthesize chlorophyll and structural proteins required for thylakoid system. In light grown seedlings the chloroplast develops directly from the protoplastids. When etiolated seedlings are exposed to light, these develop chloroplast.
If the etiolated seedlings are treated with cytokinin before they are illuminated, form chloropladt with more extensive grana. Also chlorophyll and photosynthetic enzymes are synthesized at a greater rate after illumination. These results suggest role of cytokinins in promotion of chloroplast maturation.
Stimulation of Cell Enlargement
Cytokinins can promote cell enlargement in certain tissues and organs. This effect can be noticed in dicot seeds such as those of mustard, cucumber and sunflower. The cotyledons of these species expand as a result of cell enlargement during seedling growth. Cytokinin applications promotes additional cell expansion with no increase in dry weight of the cotyledons. Cytokinin stimulate the growth of the cotyledons by increasing the plasticity of the cell walls without changing their elasticity.