The generation of tools to interrogate carbohydrate metabolism during cereal germination

Rugen, Michael (2015) The generation of tools to interrogate carbohydrate metabolism during cereal germination. Doctoral thesis, University of East Anglia.

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Starch metabolism during barley germination is important to seedling establishment and has applications in malting and brewing. There remains a lack of understanding of how this process is controlled. It has previously been shown that the iminosugar 1-deoxynojirimycin (DNJ) retards grain starch loss during germination but also causes stunted root growth in the presence of exogenous glucose, possibly by interfering with glycoprotein processing.
To analyse the effects of other iminosugars on germination, a library of 391 N-substituted DNJ analogues were screened against Arabidopsis and a monocot alternative Eragrostis tef. The most potent compound identified, N-5-(adamantane-1-yl-ethoxy)pentyl-ido-DNJ (Ido-AEP-DNJ), inhibited root growth by 92% and 89% in Arabidopsis and tef, respectively, at 10 ?M. Further analysis implicated glucosylceramide synthase as a target responsible for the effect caused by Ido-AEP-DNJ.
Effective small molecule inhibitors have previously been identified for the barley enzymes ?-amylase, ?-amylase and ?-glucosidase. The debranching enzyme limit dextrinase (LD) is the sole enzyme responsible for the hydrolysis of ?-1,6-linked dextrins during germination, to date, no potent inhibitors have been identified for LD. When assayed against LD (expressed in Pichia pastoris) the glycosylated variants of DNJ: G1M and G2M show inhibition of 80% and 90%, respectively, at 1 mM. Potential peptide inhibitors, based on the sequence of the proteinaceous LD inhibitor (LDI) were also analysed.
To enable further study, genes encoding LD and LDI were cloned and expressed as hexahistidine fusions in E. coli. Soluble LD was purified 75 fold by nickel affinity, ?-cyclodextrin affinity and size-exclusion chromatography. LDI expressed in an insoluble form, but was solubilised and purified using ?-mercaptoethanol, urea and nickel affinity chromatography. Polyclonal antibodies were raised against the recombinant proteins. Homozygous RNAi lines for knock-down of LD and LDI, alongside constructs to study subcellular localisation, were also generated to further probe the roles of these proteins in planta.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Jackie Webb
Date Deposited: 19 Jul 2016 15:31
Last Modified: 19 Jul 2016 15:31

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