Sarana Sommano (2011). BROWNING MECHANISM IN BACKHOUSIA MYRTIFOLIA PhD Thesis, School of Agriculture and Food Sciences, The University of Queensland.

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Author Sarana Sommano
School, Centre or Institute School of Agriculture and Food Sciences
Institution The University of Queensland
Publication date 2011-03
Thesis type PhD Thesis
Supervisor Prof. Daryl Joyce
Dr. Bruce D'arcy
Dr. Priya Joyce
Total pages 179
Total colour pages 23
Total black and white pages 156
Subjects 07 Agricultural and Veterinary Sciences
Abstract/Summary Backhousia myrtiflolia is an Australian native that has recently been cultivated for cut flowers. Browning in the leaves and flowers (sepals) of this plant has become a hurdle for the export marketability of its cut foliage. Understanding the mechanism and triggers for this discolouration is important for the further development of methods that could ideally prevent browning. This research study focussed on a biochemical approach to understanding the enzymatic browning process. One clear cause of browning in this species is reported herein. Browning in B. myrtifolia potted plant leaves was observed during winter 2008 - 2009. Koch’s postulates were performed with leaf lesion explants. The discolouration proved to be a result of plant disease from a single fungus morphologically identified and confirmed as Alternaria alternata. Inoculations on attached leaves and detached flowers were positive in terms of causing browning. However, not all browning in B. myrtifolia can be attributed to pathogenic infection. The physical and biochemical browning processes of B. myrtifolia were investigated. Browning was elicited by heating the tissues at 60C for 30 and 60 min. Changes in electrolyte leakage (EL), malondialdehyde (MDA) content, polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonialyase (PAL) enzyme activities, total phenolic content and antioxidant capacity (DPPH) were monitored. Leaves were separated into two types, yellow and green, due to observed differences in their browning susceptibility. They were tested for chlorophyll content (Chl a and b) and chlorophyll fluorescence (CF) prior to browning induction. Heat treatment had generally similar effects on physiological and biochemical parameters in both leaf tissue types and also sepals. However, high browning susceptibility in the yellow leaf type was associated with higher post-treatment EL, relatively high initial PPO activity and high pre- and post-treatment PAL activity. The greater initial PPO activity and higher concentrations of reactive oxygen species (ROS) evidently resulted in chlorophyll bleaching in yellow leaves. Light microscopy images of tissues before and after heat-induced browning treatment were compared. The tissues were cleared of chlorophyll pigments using choral hydrate solution. Browning was intensively found in cells surrounding gland structures identified by light and scanning electron microscopy. Browning also appeared on the surface of B. myrtifolia leaves to with B. citriodora essential oil had been applied. This observation suggested an oleocellosis-like phenomenon in B. myrtifolia. Polyphenol oxidase (PPO) and polyphenol peroxidise (POD) are two major enzymes involved in post-harvest browning reactions. Characterisation of these enzymes from B. myrtifolia tissues involved the removal of endogenous phenols by incoperating polyvinyl pyrorolidone (PVPP, an insoluble phenol-absorbing agent) at a concentration of 5% (w/v) in 0.1 M sodium phosphate buffer pH 6.8). PPOs were also activated by the use of Triton X 100 (TTX-100; 2% v/v). POD activity was similary improved by using the same extraction process as for PPO. The protein form the PPO-POD extract (also known as crude extract) was concentrated using an Amicon® filter unit and used for native electrophoresis. The concentrated protein extract lost spectrophotometric activity presumably due to the high amount of phenols and chlorophylls released after detergent was added to the extraction solution. However, strong PPO activity band was detected from this extract with native electrophoresis. Staining of gels for POD activity was not successful. For further purification of the PPO-POD extract, high temperature induce phase partitioning in TTX-114 which eliminated chlorophylls and remaining phenols and thereby improved the enzymatic activity was used. . The enzyme extract partically purified using this optimised extraction combination were characterised for their reaction to pH, their temperature optima, their substrate specificity and their reaction kinetics. The results suggested that PPO and POD from both leaf and floral tissues were of different isozymes as there were peaks in activities in both acidic and basic pH regions. PPO and POD from both tissues types were heat sensitive with temperature optima at 20 - 25°C. Both enzymes were active with mono and diphenol substrates. A kinetic study of these enzymes was made using the partially purified extracts. The enzymatic rate constant (Km) for both PPO and POD suggested that the intercellular substrate concentration was higher in floral tissues than in leaf tissues. It also confirmed that PPO and POD from leaf and floral tissues were different isozymes. The effects of inhibitors of PPO and POD from B. myrtifolia leaf and floral tissues were also investigated. Glutathione was an uncompetitive inhibitor to PPO in leaf and floral tissues at a high concentration of 10 mM. Soduim azide at 0.005 - 0.5 mM was competitive to POD from both leaf and floral tissue types. Enzyme localisation was also examined by using a subcellular extraction method. PPO and POD from both B. myrtifolia leaf and flower tissues were mainly membrane bound enzymes as detected by native electrophorersis (PPO) and Western analysis with specific antibodies (POD). Multiple isoforms were confirmed by 2 dimensional electrophoresis (2 DE) patterns. Overall, browning in B. myrtifolia leaf and floral tissues was a result of cell compartment degeneration which was triggered by stresses (i.e., heat, essential oil toxicity and pathogen). Thereafter, browning related enzymes namely PPO, POD, PAL-phenol reactions were active. Results also suggested that browning was associated with damage to photosynthesis apparatus when plants were grown in the fully-opened area during severe winter condition. Both B. myrtifolia leaf and foral tissues contain a considerable high amount of polyphenol content. This together with high chlorophyll pigments have to be eliminated prior to PPO and POD characterisation study. PPO and POD from leaf and floral tissues are membrane bound proteins and presented in different isozymes as well as different isoforms.
Keyword Australian native flower, browning mechanism, characterisation, discolouration, enzyme extraction, enzyme kinetic, export market, infection, oleocellosis, postharvest
Additional Notes 21,23,24,32,36,69,70,71,80,83,91,106,109,116,126,138,139,161,162,163,164,172,173

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Created: Sat, 13 Aug 2011, 08:12:27 EST by Mr Sarana Sommano on behalf of Library - Information Access Service