Carbon Mobilisation and Utilisation in the Sugarcane Biofactory

Brian O'Neill (2011). Carbon Mobilisation and Utilisation in the Sugarcane Biofactory PhD Thesis, School of Chemical Engineering, The University of Queensland.

       
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Author Brian O'Neill
Thesis Title Carbon Mobilisation and Utilisation in the Sugarcane Biofactory
School, Centre or Institute School of Chemical Engineering
Institution The University of Queensland
Publication date 2011-10
Thesis type PhD Thesis
Supervisor Lars Nielsen
Stevens Brumbley
Matt Purnell
Total pages 108
Total colour pages 6
Total black and white pages 102
Subjects 03 Chemical Sciences
Abstract/Summary Heterotrophic axillary bud outgrowth from sugarcane (Saccharum spp. hybrids) setts was used as a model system to demonstrate that sucrose is a mobilisable carbon source. The outgrowth and subsequent biomass accumulation of axillary buds from two-eye setts of mature sugarcane stalks grown in the dark was used to measure carbon mobilisation from sett internode pith tissue. After 42 days growth 99.0 ± 0.72% of sett internode pith sucrose was depleted and 2.66 ± 0.16g of new tissue accumulated. Comparison with a control treatment in which axillary buds were excised at day zero demonstrated that carbon mobilisation was driven by the accumulation of new biomass. Profiling of soluble carbohydrates (viz. sucrose, glucose and fructose), starch, total soluble protein, total amino nitrogen, free amino acids and total insoluble material showed that the sucrose stored in the sett internode pith was the only available carbon source of sufficient size at day zero for the observed biomass accumulation. Other metabolites mobilised were glucose, fructose and some amino acids, notably isoleucine and leucine that were depleted in shoot treatment setts at day 42. Because sucrose stored in mature stalks (in excess of 40 % of stalk dry weight) can be wholly mobilised to supply carbon for the growth of heterotrophic tissues we propose that sucrose mobilisation requires a net sink-to-source transition that acts in toto within sett internode storage parenchyma. From our data it is proposed that mobilisation of sucrose from culm storage parenchyma requires minimal investment of metabolic resources and that the mechanism of sucrose mobilisation is metabolically neutral. Using magnetic resonance spectroscopy and phloem-specific tracer dyes, strong evidence is provided that sucrose is mobilised from sett storage parenchyma via phloem to the growing shoot tissue. Analysis of enzyme activities involved in sucrose metabolism and glycolysis suggest that sucrose synthase activity is down-regulated due to the effects of sucrose mobilisation. Overall, metabolism in storage parenchyma shifts from futile cycling to a more quiescent state during sucrose mobilisation. Trehalose metabolism in sugarcane was engineered in an attempt to create a significant carbon drain of stored sucrose, to impart value added properties and to enhance abiotic stress tolerance. Two transgenes were introduced to the genome: Trehalose-6-phosphate synthase/phosphatase (TPSP) to increase trehalose biosynthesis and an RNAi transgene specific for trehalase to abrogate trehalose catabolism. Plants phenotypically indistinguishable from the wild type plants were recovered that harboured each transgene singularly but no events of co-integration were observed in a total of 14 transgenic lines recovered after bombardment with both constructs. Soluble carbohydrate content was unaffected. Expression of trehalase was abrogated in many lines due to the RNAi transgene; however, no decrease in trehalase activity was observed. A trehalase inhibitor, sufficient to cease porcine trehalase activity wholly, decreased trehalase activity in sugarcane pith tissue extracts by 80%. Trehalose biosynthesis activity in TPSP lines was elevated compared to RNAi lines and the difference bordered on significance (P = 0.080). A positive correlation between TPSP and trehalase activity was present in RNAi transformants that detectably expressed trehalase (Pearson correlation co-efficient = 0.720, P = 0.107). We discuss the implications for engineering trehalose metabolism in terms of an embryonic lethal phenotype due to altered trehalose metabolism.
Keyword carbon source-sink
carbon partitioning
cagnetic resonance spectroscopy
Saccharum
sucrose mobilisation
sugarcane biofactory
alternative products
sucrose derivatives
trehalose
trehalase
Additional Notes Colour pages: 25, 29, 49, 51, 54, 62 Landscape page: 27

 
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Created: Wed, 12 Oct 2011, 10:42:14 EST by Mr Brian O'neill on behalf of Library - Information Access Service