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Cell division is a fundamental process of living meristematic tissues. Growing cells in sterile culture was of interest to early cell biologists so that one might study cellular processes without the influences of tissues, organs, and so on. The idea that the cell cycle could be regulated by chemicals was inspired by the relationship between Agrobacterium tumefaciens (a bacterium) and its host plants. An infection with this bacterium caused a rapidly growing tumor to develop in just about any tissue of suitable host species. This fact indicated that the cells of higher plants are totipotent (capable of becoming meristematic...changing its developmental fate). This tumor was a lump of undifferentiated (having no particular fate) cells. Once infected, the lump could be cured of its infection by either heat shock or by antibiotics and the tumor would continue to grow in a tumor form.
It was soon discovered that auxins could initiate the formation of callus in plant tissues too. You observed some callus at the stumps where you applied the auxin, IBA, at 5000 ppm on stems of kidney bean plants. Under good circumstances this can also initiate root formation, demonstrating that dedifferentiated callus tissue can be hormonally induced to differentiate along the line leading to root formation. You also have done a rooting project with Mung beans in which the hypocotyl increased in diameter with callus development, and then nearly burst-open with root development along the vascular traces in the stem. People trying to get tissues to grow in vitro, knew the importance of minerals and vitamins in the medium. They tried various additives to get tissues to grow optimally. Single tissues could grow for at least a limited time, but getting a whole plant to develop from the culture was not possible. One additive that seemed the help a lot was the addition of coconut milk (liquid endosperm). With this one addition and some small amount of auxin, one could regenerate an entire plant from just about any tissue. The search was on to find out what the "magic" of the coconut milk was. Just recently (2001) Folke Skoog died...he and his colleague Carlos Miller tried many possible substitutes for coconut milk. The goal was to find out what chemical could stimulate cell division and be far more reliable that the batch-to-batch inconsistency observed with coconut milk. The nitrogenous base, adenine, had at least some activity in this regard. Fresh herring sperm DNA was totally inactive, but autoclaved herring sperm DNA stimulated the cell cultures. They purified the fractions from this sample until they had a chemical that worked...they named it kinetin...and determined its chemical structure in 1955.
Indeed you can see that this is an adenine derivative (6-furfurylaminopurine). In spite of this, kinetin is not a natural cytokinin. Many nitrogenous bases are modified in various ways in the DNA of organisms, but this particular chemical has never been found to occur without the autoclaving manipulation. This discovery led researchers to start looking for other compounds that would be active in cell division. The concept was based on the idea of structure-activity relationship. Obviously the natural cytokinin must have structure similar to kinetin. Simultaneously the pharmaceutical industry started screening synthetic compounds that would be even more effective than kinetin. The results of those studies are shown above. As you might recall we have been using kinetin and benzyl adenine in various projects in lab. I think you recall from the tissue culture project that we got excellent shoot development in media with some BA and could get excellent callus with a different ratio of BA:NAA. Indeed the synthetic cytokinin, BA, is used routinely in tissue culture as it is stable to autoclaving and therefore is easy to use in such work. The antagonist discovered has been thought to be a competitive inhibitor for the receptor for cytokinins in plant cells...another structure-activity relationship.
Again you will notice how these are adenine derivatives and these can be part of a nucleotide (with added ribose sometimes with the phosphate too). The most common natural cytokinin in plants is trans-zeatin. A graph showing the dose responses of tobacco callus cultures to zeatin and kinetin shows that the callus tissue is far more sensitive to the natural zeatin than it is to the synthetic kinetin...you did this project in class...were your results the same or different?
You will notice above that the first steps of the pathway are known only vaguely in plants! But as the genome of Arabidopsis has been searched, more of the enzymes are being found by comparison with bacterial genes. The pathway continues, ultimately producing IPA and Zeatin. I am assuming here that you know how plants produce five-carbon sugars or five-carbon sugar phosphates to do this. That IS a valid assumption, right?!
However, it is also true that the ribosides themselves are a form of conjugation. All studies to date seem to indicate that the free-base has to be cleaved from the ribose too before the compound has any true activity. Plants carry out this reaction easily and rapidly, so ribosides appear to have activity on their own, but this is an artifact. Cells in culture require the cytokinin to be free...these sometimes lack the enzymes to cleave the ribose, so supplied ribosides are inactive and the free-base must be supplied in the medium. The native cytokinins also occur as modified bases in RNA and DNA strands. In fact cis-zeatin (a less active form) is found in many tRNA molecules in almost all living cells of all species! The extent to which the free cytokinin pool is altered by conjugation with other nucleotides or released from nucleotide polymers is not clear.
The Agrobacterium injects a plasmid (naked circular DNA) into the host (in this case tomato) cells. This plasmid is called the Ti (Tumor inducing) plasmid. This piece of prokaryotic DNA has two segments of DNA called the "left border" and the "right border" with genes in between. These "borders" permit recombination of the genes into the host genome. The genes turn on cytokinin synthesis! The structure of the T-DNA in the Ti plasmid is linearized below...
This, in turn results in the development of a "crown gall" tumor on the plant. The fact that the plant can be cured of the bacteria later and the tumor continues to thrive, simply demonstrates that the Ti DNA has become a permanent addition to the host genome. The bacteria may be killed, but the genes remain in the cells. This realization of course provided the smart researcher with a useful tool for transforming plant cells with foreign DNA. Anytime a scientist wants to insert an engineered gene into a plant cell, the gene simply has to be put between the borders of the Ti plasmid (probably along with an antibiotic resistance gene for selection purposes) and let the Agrobacterium inject the gene into the cells for incorporation in the genome. Of course we are interested in making a transgenic whole plant, so we cut out the cytokinin synthesis gene and replace it with the gene of our research interest. Since the cytokinin over-production genes are absent, the cells can develop into whole transgenic plants (thanks to totipotency of plant cells!). This process is briefly outlined below. The relative amounts of cytokinins and auxins can regulate the differentiation that can occur from transformed cells. So when it is time to regenerate whole plants from engineered cells, the relative ratio of these two hormones regulates what develops. Here you can see an array of cytokinin concentrations and auxin concentrations on callus growth. The no hormone control is in the lower left corner.
A close-up of the four most-interesting of those plates is presented below. Here you can see the combinations of hormones that control differentiation. Explants just increase in size but maintain leaf morphology in the control. With lots of both cytokinin and auxin, callus proliferates as in the normal Crown Gall form as caused by the normal Ti-plasmid from Agrobacterium. With low cytokinin but lots of auxin you get rooty explants which is what you noticed in our Mung-bean lab exercise. With low auxin but lots of cytokinin the explants form callus and then produce small shoots. Moving transformed cells among these four hormone concentrations, it is possible to regenerate whole cells from them.
Here you can see in seedling B that the cytokinin-treated leaf on the left attracted the radio-tagged amino acid from the untreated leaf on the right. In seedling C, when the tagged leaf is also treated with cytokinin, there is not even the small amount of leakage to the other leaf observed in the control (seedling A).