METABOLISM OF PLANTS

The diamine putrescine, the triamine spermidine and the tetramine spermine occur as free cations and as conjugates with phenolic acids and macromolecules in plants. Their levels of concentration increase greatly when the environment suffer changes; specially when there is potassium deficiency, water deficits, salinity stress, anaerobiosis and acid stress ("Polyamines"). Because polyamines are synthesized by amino acid decarboxylation reactions, where H+ is consumed, levels of polyamines may function as a protection method; that is haw they conserve the pH values constant ("Polyamines"). Polyamines are alsoprecursors of various alkaloids that are important in plants defense.

 
The first polyamine formed is putrescine. This may happen by decarbosylation of ornithine or arginine.  These reactions are catalyzed by the enzymes orthinine decarboxylase (ODC) or arginine decarboxylae (ADC), respectively. The polyamines spermidine and spermine are formed by adding an aminopropyl moeity to putrescine and spermine, respectively.

Polyamines can occur as free molecular bases in plant cells, but they also occur as conjugates, associated with small molecules like phenolic acids and macromolecules such as proteins.  Polyamines are most commonly conjugated to cinnamic acids, preferentially p-coumaric, ferulic and caffeic acids, and they occur in a wide range of plant families. 

Adequate levels of polyamines are necessary for optimal growth and replication of plants, bacteria and fungi.("Polyamines")

Polyamine Metabolism and Ionic Stress:

1. Potassium deficiency:
Plants undergo great changes in polyamine's levels, especially Putrescine levels, as a result of nutritional deficiencies.  Previous works have shown that Putrescine accumulates in K+ deficient leaves of tomato, cabbage, radish, lettuce, pea, beet, oat, rye, tobacco, banana, maize, Ribes nigrum, broad bean, and spinach. Therefore, diamine accumulation as a result of potassium deficiency is widespread among mono and dicotyledonous plants. Putrescine in K+ deficient barley and clover can reach levels of 23 µmol/g dry weight. Putrescine accounts for at least 20 % of the total plant nitrogen under these circumstances (Slocum 1991).

2. Other mineral deficiencies:
Deficiency in other minerals and salts such as K, P, Ca, Mg, Fe, Mn, S, B and KCl also cause increase in the levels of polyamines, but not in such a striking range as K+ (Slocum 1991).

3. Acid stress:
Under acidic conditions, ADC activity increases. The amines produced by this, help raise the medium pH to values more favorable to the growth(Slocum 1991).

1. Environmental pollutants:
Ozone is an important component of air pollution. It has significant influences in photosynthesis. Levels of Ozone induce a number of biochemical changes. One of these changes is the increase in polyamines(Slocum 1991).

2. Heat and drought stress:
The usual response to stress is an increase in one, or sometimes more, of the polyamines that was already there before the stress. However, under heat stress, it has been shown that plant cells can induce the formation a new polyamines.  The most common of these polyamines formed under heat stress are norspermidine and norspermine (Slocum 1991).

Assay of enzyme activities ( ADC, ODC and SAMDC ) (Burtin and Michael):

• Frozen leaf powder is homogenized in 0n1 M Hepes buffercontaining 10 mM dithiothreitol, 10 mM EDTA, 0n1 mM pyri-doxal 5h-phosphate and 0n5% (w\v) ascorbic acid.
• Insolublepoly(vinyl pyrrolidine) (100 mg\g of powder) is added beforehomogenization.
• The homogenates are  centrifuged for 25 minat 17000g and the supernatants loaded on to a PD10 column (Pharmacia) and eluted with the same buffer minus the ascorbicacid.
• The eluates are used for analysis of enzyme activity
• Enzyme reaction mixtures contains 100 µl of extract and 20 µl of radiolabelled substrate
• The ODC assay mixture contains 0n1 µCi of -[1-14C]ornithine (57 mCi\mmol) and unlabelledornithine (2n5 mM). -[U-14C]arginine (0n1 µCi, 307 mCi\mmol) and unlabelled arginine (1n25 mM final concentration) are added for the analysis of ADC activity.
• For SAMDC activity, 20 µl of 5 µCi\mlS-adenosyl- -carboxy[14C]methionine dilutedin 4n5 mM unlabelled SAM is used.
• Each reaction is performed in duplicate at 37 mC for 45 min
• The ADC, ODC andSAMDC activities are determined by measurement of 14CO2 released from substrates

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