טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMor Meital
SubjectIn Vitro Effects of Boron on Enzyme Reactions involved in
Lignin Synthesis
DepartmentDepartment of Civil and Environmental Engineering
Supervisors Professor Emeritus Peter Neumann
Professor Emeritus Carlos Dosoretz
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Boron is widely distributed throughout the Earth’s land and water. Relatively high concentrations are often found in dry terrains, such as in the Negev desert and Jordan valley in Israel. Boron is an essential plant micro-element that is absorbed from soil water by the roots.

Boron concentrations in soils can be increased when crops are irrigated with recycled waste water. Excessive accumulation of boron in plants leads to growth inhibition.

The functioning of boron in plant (and animal) cells is related to its ability to form complexes with cis-diols on sugars, and phenolic compounds such as caffeic acid. Caffeic acid is an essential precursor in the complex lignin synthesis pathway. Deposition of phenolic cross links and lignin in plant cell walls decreases wall extensibility and inhibits cell growth. A central hypothisis investigated in this research was that high cellular boron levels might increase lignin biosynthesis, and thereby reduce plant growth.

In vitro effects of boron complexation with caffeic acid on enzymatic reactions involved in lignin biosynthesis were therefore investigated. To the best of our knowledge, the possible metabolic effects of any interactions between boron and the cis-diol groups of caffeic acid have not been investigated. 


Caffeic acid methylation to ferulic acid by caffeic acid methyltrasferase (COMT) was not affected by the presence of boron. The methylation produced meta and para ferulic acid and there was no change in the reaction rate or in the amount of caffeic acid that was transformed to ferulic acid. The enzyme showed little or no activity at pH=5, the pH level found in cell walls. Thus, it is likely that the methylation occurs in the cytoplasm at pH=7 rather than in the more acidic cell walls.


Caffeic acid and ferulic acid oxidation catalysed by horseradish peroxidase (HRP) and tyrosinase resulted in phenolic radicals that produced caffeic acid and ferulic acid polymers. The polymers were analyzed by mass spectroscopy and gel filtration HPLC in order to determine their molecular masses and consequently the number of caffeic acid monomers included in them.    

It was found that the phenolic polymers produced in the presence of high boron concentrations (0.4 mM) had higher molecular weights than those produced in the absence of boron. Moreover, the amount of substrate utilized was the same regardless of boron concentration. Therefore, it can be concluded that excess boron affected the degree of polymerization.