Abstract
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.
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Claims 19
Claim · model-written summary
Sett internode pith sucrose was 99.0% depleted after 42 days of bud outgrowth.
Verbatim
t0“Sugarcane Biofactory”
e1“After 42 days growth 99.0 ± 0.72% of sett internode pith sucrose was depleted and 2.66 ± 0.16g of new tissue accumulated”
Claim · model-written summary
Axillary bud outgrowth accumulated 2.66 g of new tissue after 42 days.
Verbatim
t0“Sugarcane Biofactory”
e2“2.66 ± 0.16g of new tissue accumulated”
Claim · model-written summary
Carbon mobilisation was driven by the accumulation of new biomass, as shown by comparison with excised buds.
Verbatim
t0“Sugarcane Biofactory”
e3“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”
Claim · model-written summary
Stored sucrose was the only available carbon source of sufficient size at day zero for the biomass accumulation.
Verbatim
t0“Sugarcane Biofactory”
e4“the sucrose stored in the sett internode pith was the only available carbon source of sufficient size at day zero”
Claim · model-written summary
Isoleucine and leucine were depleted in shoot treatment setts at day 42.
Verbatim
t0“Sugarcane Biofactory”
e5“notably isoleucine and leucine that were depleted in shoot treatment setts at day 42”
Claim · model-written summary
Sucrose mobilisation is proposed to require a net sink-to-source transition in the storage parenchyma.
Verbatim
t0“Sugarcane Biofactory”
e6“we propose that sucrose mobilisation requires a net sink-to-source transition that acts in toto within sett internode storage parenchyma”
Claim · model-written summary
Sucrose mobilisation from culm storage parenchyma is proposed to require minimal metabolic investment.
Verbatim
t0“Sugarcane Biofactory”
e7“From our data it is proposed that mobilisation of sucrose from culm storage parenchyma requires minimal investment of metabolic resources”
Claim · model-written summary
The mechanism of sucrose mobilisation is proposed to be metabolically neutral.
Verbatim
t0“Sugarcane Biofactory”
e1“the mechanism of sucrose mobilisation is metabolically neutral”
Claim · model-written summary
Sucrose is mobilised from sett storage parenchyma via phloem to the growing shoot tissue.
Verbatim
t0“Sugarcane Biofactory”
e1“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”
Claim · model-written summary
Sucrose synthase activity is down-regulated by sucrose mobilisation.
Verbatim
t0“Sugarcane Biofactory”
e1“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”
Claim · model-written summary
Storage parenchyma metabolism shifts from futile cycling to a more quiescent state during sucrose mobilisation.
Verbatim
t0“Sugarcane Biofactory”
e1“Overall, metabolism in storage parenchyma shifts from futile cycling to a more quiescent state during sucrose mobilisation”
Claim · model-written summary
Transgenic plants carrying each transgene singly were phenotypically indistinguishable from wild type.
Verbatim
t0“Sugarcane Biofactory”
e1“Plants phenotypically indistinguishable from the wild type plants were recovered that harboured each transgene singularly”
Claim · model-written summary
No co-integration events were observed among 14 transgenic lines.
Verbatim
t0“Sugarcane Biofactory”
e1“no events of co-integration were observed in a total of 14 transgenic lines recovered after bombardment with both constructs”
Claim · model-written summary
The RNAi transgene abrogated trehalase expression in many lines.
Not reportedComparator
Verbatim
t0“Sugarcane Biofactory”
e1“Expression of trehalase was abrogated in many lines due to the RNAi transgene”
Claim · model-written summary
No decrease in trehalase activity was observed in the RNAi lines.
Verbatim
t0“Sugarcane Biofactory”
e1“no decrease in trehalase activity was observed”
Claim · model-written summary
A trehalase inhibitor decreased trehalase activity in sugarcane pith extracts by 80%.
- Taxon
- Sugarcanet0
- Organ / stage
- pith tissue extractse1
- Treatment
- trehalase inhibitore1
- Outcome
- trehalase activity in sugarcane pith tissue extractse1
- Direction
- decreasede1
- Value
- 80%e1
Not reportedComparator
Verbatim
t0“Sugarcane Biofactory”
e1“A trehalase inhibitor, sufficient to cease porcine trehalase activity wholly, decreased trehalase activity in sugarcane pith tissue extracts by 80%”
Claim · model-written summary
Trehalose biosynthesis activity was elevated in TPSP lines compared with RNAi lines, borderline significant.
Verbatim
t0“Sugarcane Biofactory”
e1“Trehalose biosynthesis activity in TPSP lines was elevated compared to RNAi lines and the difference bordered on significance (P = 0.080)”
Claim · model-written summary
Trehalase activity was positively correlated with TPSP in RNAi transformants that expressed trehalase.
Verbatim
t0“Sugarcane Biofactory”
e1“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)”
Claim · model-written summary
Soluble carbohydrate content was unaffected by the transgenes.
Verbatim
t0“Sugarcane Biofactory”
e1“Soluble carbohydrate content was unaffected”