The characterisation of nascent pectin complexes in pea plants

Rizkallah, Hind Dunyah (2005) The characterisation of nascent pectin complexes in pea plants. PhD thesis, University of Glasgow.

Abstract

The structure and properties of a nascent pectin-xyloglucan complex in etiolated pea epicotyls were investigated. Membrane pellets were prepared and incubated with UDP-[14C] galactose, and were extracted with six different reagents: Tris-HC1 buffer (pH 7.4); 50mM EDTA/50mM PO4 (pH6.8) at 100°C; 50mM EDTA/59mM PO4 (pH6.8) at 25°C; phospholipase C with 100 ml of 0.1M Tris-HC1 pH7.4 at 25°C; trypsin at 25°C and 0.1% Triton X-100 at 25°C. The best extractant used to solubilise the pectin-complex from the pellets was 50nM EDTA/50mM PO4 pH 6.8 at 100°C. Aqueous solutions of pectic polyuronides tend to associate covalently into multichain aggregates. Because of the tendency of pectins to aggregate in solution, the effect of a number of eluents on the behaviour of the complex on gel filtration was studied. 10mM EDTA/10nM PO4/1M NaC1 (pH6.8) was chosen as the best eluent to minimise this aggregate formation. On gel filtration using Sepharose CL-2B with this eluent, the complex eluted with a Kav of around 0.8, corresponding to a molecular size approximately 200 kDa, as judged by dextran standards. In other solvents tested, aggregation appeared to occur. On Sepharose CL-4B and CL-6B columns using the same EDTA/Pi/NaC1 solvent, the apparent molecular size was significantly reduced by a xyloglucan-specific endoglucanase, confirming the presence of a xyloglucan rather that a glucan in the complex. Polygalacturonase caused a greater decrease in apparent molecular size, to 10-20 kDa while endo-1 4-b-galactanase converted the radioactivity to [14C] galactobiose and [14C] galactose. When the [14C] galactose-labelled complex was incubated in solution with 3MM paper, the radioactivity was almost completely absorbed onto the paper over a period of about 6 hours. This confirmed the presence of xyloglucan attached to pectin. A new model for the structure of the nascent pectin-xyloglucan complex is proposed.

• Item Type: Thesis (PhD)
• Qualification Level: Postdoctoral
• Subjects: Q Science > QK Botany
• Colleges/Schools: College of Science and Engineering
• Supervisor's Name: Brett, Doctor Chris
• Date of Award: May 2005
• Depositing User: Mrs Marie Cairney
• Unique ID: glathesis:2005-7092
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 11 Feb 2016 14:33
• Last Modified: 22 Apr 2016 11:00
• URI: https://theses.gla.ac.uk/id/eprint/7092