Plant Physiology (Biology 327)  - Dr. Stephen G. Saupe;  College of St. Benedict/ St. John's University;  Biology Department; Collegeville, MN  56321; (320) 363 - 2782; (320) 363 - 3202, fax;    ssaupe@csbsju.edu

Plant Hormones - Cytokinins

I. General

• Called "cytokinins" because they stimulate cell division (i.e., cytokinesis)
• Haberlandt (1913) noted that non-dividing potato parenchyma cells would revert to actively dividing ones in the presence of phloem sap.  This observation suggested a soluble material was responsible for cell division. 
• Folke Skoog (1940's) and colleagues at Univ. of Wisconsin found that cultured tobacco pith tissue explants would proliferate only if they were supplemented with various substances such as autoclaved herring sperm or coconut milk.
• Miller (1956) identified the first cytokinin, called kinetin, in the herring sperm.
• Cytokinins occur in most plants including mosses, ferns, conifers, algae and diatoms

II. Chemistry

A. General

• adenine derivatives (amino purines)
• occur as: (a) the free nitrogenous base; (b) a nucleoside (base + ribose); (c) a nucleotide (base + ribose + phosphate); or (d) glycosides
• The free base is the active form.
• approximately 40 different structures known.
• Zeatin (Z), which was first isolated from maize (Zea mays) is the most common cytokinin.
• Other naturally occurring cytokinins include, dihydrozeatin (DHZ) and isopentenyladenosine (IPA).

B. Synthetic cytokinins

• kinetin (as above) – probably byproduct of zeatin degradation
• there are several other substances with cytokinin activity such as benzyl adenine (benzylaminopurine; BA).

C. Cytokinins and nucleic acids

• can occur as a modified base in tRNA, but the bases exist in the cis form, rather than the typical trans form. These modified bases that are found in all organisms from bacteria to plants to humans.
• The function of the tRNA cytokinins is not clear, but after hydrolysis of the tRNA the products can act as a cytokinin. The importance of the tRNA derived cytokinins in overall growth and development is not clear, either.
• Interestingly plants have different sets of tRNA’s with different cytokinins that participate in protein synthesis in the cytoplasm and the plastids.

III. Synthesis

• Site:  synthesized primarily in the meristematic region of the roots.  This is known in part because roots can be cultured (grown in artificial medium in a flask) without added cytokinin, but stem cells cannot.  
• Cytokinins are also produced in developing embryos and crown gall tissues
• first major precursor to the cytokinin is AMP (adenosine monophosphate)
• side chains of the cytokinins are made by the terpene pathway (IPP – isoprene) and added to the AMP by cytokinin synthase
• there is some speculation that surface and endophytic bacteria (i.e., Methylobacterium) may be the actual source of plant cytokinins.  Rationale:  (1) haven't isolated some of the putative genes for cytokinin synthesis; (2) remove bacteria show impaired cytokinin production.
• tRNA nucleotides are modified to cytokinins after the tRNA is transcribed (post-transcriptional processing)

• via xylem (transpiration stream)
• in peas, a signal from the leaves may signal/regulate transport of cytokinins from the roots
• zeatin ribosides are the main transport form; converted to the free base or glucosides in the leaves
• some cytokinin also moves in the phloem.

V. Bioassays/Analysis

A. Bioassay

1. Callus culture cell proliferation - not used too much because it takes too long
2. Expansion of radish or cocklebur cotyledons
3. Inhibition of chlorophyll loss by detached oat leaves during senescence

B. Methods of Analysis – liquid chromatography, mass spectroscopy; radioimmunoassays

VI. Disposal

• forms conjugates with glucosides. major storage form of cytokinin, inactive
• cytokinin oxidase may be an important route of disposal, too.

VII. Actions

A. Control morphogenesis

• in plant tissue cultures, cytokinin is required for the growth of a callus (an undifferentiated, tumor-like mass of cells):

• 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

• some tissues become habituated during repeated cell culture – loose the requirement for cytokinin in the growth medium

B. Crown Gall

• tumor-like mass of undifferentiated cells that typically occurs near the crown (junction of root and stem) of the plant
• caused by the bacterium Agrobacterium tumefaciens
• carries a plasmid (Ti plasmid; a plasmid is a small loop of non-chromosomal DNA) with loci/genes for auxin production (tms), zeatin production (tmr) and opines (are nitrogen-containing molecules that provide food for the bacteria).
• upon infection, the plasmid is incorporated into the plant cell genome which begins to overproduce auxin and cytokinin
• stem forms an undifferentiated tumor (crown gall)
• as predicted, if the tms genes (auxin production) are deleted from the plasmid, which would increase the cytokinin/auxin ratio, the resultant crown gall is "shooty". If the tmr genes are deleted the gall is "rooty".

C. Regulates the cell cycle/cell division (hence, the name "cytokinins) – especially by controlling the transition from G2 � mitosis. This effect is moderated by cyclin-dependent protein kinases (CDK's) and their subunits, cyclins.

D. Delay senescence

• senescence is the programmed aging process that occurs in plants (and other organisms for that matter).
• loss of chlorophyll, RNA, protein and lipids.
• cytokinin application to an intact leaf markedly reduces the extent and rate of chlorophyll and protein degradation and leaf drop
• correlation between cytokinin levels and senescence. For example, as detached leaves senesce the cytokinin levels drop. And, when these leaves are treated with auxin to stimulate rooting, when roots form the senescence process stops and cytokinin levels rise.
• Tobacco plants + senescence promoter sequence + ipt gene (cytokinin gene) � causes gene to become active on senescence � no senescence

The exact mechanism by which this occurs is unclear but likely involves the ability of cytokinins to mobilize nutrients. Application of cytokinin to a leaf will cause it to act as a sink and nutrients will be directed towards it.

E. Greening
    Promotes the light-induced formation of chlorophyll and conversion of etioplasts to chloroplasts (greening process).

F. Promote lateral bud development
    Cytokinin application to dormant buds will cause them to develop. A witches’ broom is caused by a pathogen such as the bacterium Corynebacterium fascians (or A. tumefaciens) that produces cytokinin which, in turn, causes stimulates lateral bud development (branching). These results suggest that apical dominance may be related to cytokinin, too.

    For example, when tobacco cells are infected with the Ti-plasmid that has been modified to possess the heat shock promoter, a heat treatment stimulates the cells to produce increased amounts of cytokinin. These plants exhibit less apical dominance and remain green longer than non-heat treated controls. Thus, these results support the conclusion that senescence and apical dominance are related to cytokinin levels.

G. Promote cell expansion
     Cytokinins stimulate the expansion of cotyledons. This is the basis for the classical bioassay. The mechanism is associated with increased plasticity of the cell wall, not associated with acidification.

VIIII. Mechanism of action - Specific binding sites (receptor) for cytokinin are known. These may be ribosomal proteins. Thus, it is not too surprising that cytokinins have been shown to regulate protein synthesis.