Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2005. / The current understanding of the regulation of sucrose accumulation is still incomplete even
though many scientists have investigated this subject. Components of trehalose metabolism have
been implicated in the regulation of carbon flux in bacteria, yeast and more recently in plants. With
a view to placing trehalose metabolism in the context of cytosolic sugarcane sucrose metabolism
and carbon partitioning we have investigated the metabolites, transcripts and enzymes involved in
this branch of carbohydrate metabolism in sugarcane internodal tissues.
Sugarcane internodal trehalose levels varied between 0.31 ± 0.09 and 3.91 ± 0.99 nmol.g-1 fresh
weight (FW). From statistical analysis of the metabolite profile it would appear that trehalose does
not directly affect sucrose accumulation, although this does not preclude involvement of trehalose-
6-phosphate in the regulation of carbon partitioning. The metabolite data generated in this study
demanded further investigation into the enzymes (and their transcripts) responsible for trehalose
metabolism.
Trehalose is synthesised in a two step process by the enzymes trehalose-6-phosphate synthase
(EC 2.4.1.15, TPS) and trehalose-6-phosphate phosphatase (EC 3.1.3.12, TPP), and degraded
by trehalase (EC 3.2.1.28). Two novel sugarcane partial cDNAs that coded for trehalase (tre) and
actin (required for normalisation in profiling experiments) were isolated and used along with partial
transcripts for TPS and TPP to determine transcript levels in different tissue- and genotypes. A
putative full-length SugTPS cDNA was isolated and characterised. Enzyme activities for TPS, TPP
and trehalase were measured at levels of 2.7 nmol.min-1.mg-1protein, 8.5 nmol.min-1.mg-1protein
and 6.2 nmol.min-1.mg-1protein respectively, from young internodal protein extracts of sugarcane,
variety N19. TPP enzyme activity and transcript levels were higher in S. spontaneum than
Saccharum interspecific hybrids.
Kinetic analysis of TPP and trehalase activities were performed with the purpose of providing
parameters for an in silico kinetic model of trehalose and sucrose metabolism. Three isoforms of
TPP were identified and desingated TPPAI, TPPAII and TPPB. Both TPPA isoforms had pH
optima of 6.0, and TPPB of pH 6.5. Apparent Km values were determined as 0.447 ± 0.007 mM for
TPPAI, 13.82 ± 1.98 mM for TPPAII and 1.387 ± 0.18 mM for TPPB. Partial purification and
characterisation of trehalase demonstrated dual pH optima of 3.5 and 6.0, with Km values between
0.345 and 0.375 mM. These data were used as the basis for a kinetic model of trehalose
metabolism.
A previously described kinetic model of cytosolic sucrose metabolism has been expanded to
include the trehalose pathway (TPS, TPP and trehalase). The aim was to supplement the
available information on cytosolic metabolism in sugarcane storage parenchyma, identify points of
control between sucrose and trehalose metabolism, and provide a platform from which further
experimental and in silico modelling can be launched. The model predicted trehalose in the same
order of magnitude as those determined in the metabolite profiling experiments. The majority of
control of flux over the trehalose pathway resided in the TPS step, with flux control coefficients >
70% of the total pathway. Incorporation of the trehalose branch into the original sucrose model
showed that reactions from the original model significantly affected the steady-state attributes of
the trehalose pathway.
Due to the relatively low flux through the trehalose branch of the expanded model, complete
recycling of trehalose, and the lack of allosteric regulation by trehalose-6-phosphate or trehalose
on any of the reactions from the original sucrose model, incorporation of the trehalose branch had
no significant effect on either steady-state cytosolic sucrose concentration or flux of sucrose into
the vacuole. The expanded model affords a basis from which to further investigate trehalose
metabolism in the context of plant sucrose accumulation.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1433 |
| Date | 12 1900 |
| Creators | Bosch, Susan |
| Contributors | Botha, F. C., Rohwer, J. M., University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB) |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 2963391 bytes, application/pdf |
| Rights | University of Stellenbosch |
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