Carbon turnover and sucrose metabolism in the culm of transgenic sugarcane producing 1-Kestose
Date
2007-12
Authors
Nicholson, Tarryn Louise
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : University of Stellenbosch
Abstract
Carbon partitioning was investigated in sugarcane (Saccharum spp. hybrids) that was
genetically modified with sucrose: sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99)
from Cynara scolymus. This enzyme catalyses the transfer of a fructosyl moiety from
one sucrose molecule to another to produce the trisaccharide 1-kestose. Molecular
characterisation of four sugarcane lines, regenerated after transformation, confirmed
that two lines (2153 and 2121) were transgenic, with at least one intact copy of 1-SST
present in line 2153, and a minimum of five copies (or portions thereof) present in line
2121. The novel gene was successfully transcribed and translated in both lines, as
confirmed by cDNA gel blot hybridisation and HPLC analysis respectively.
Kestose production was stable under field resembling conditions and levels of this
trisaccharide progressively increased with increasing internodal maturity from 7.94 ±
2.96 nmol.g-1 fresh mass (fm) in internode 6 to 112.01 ± 17.42 nmol.g-1 fm in internode
16 of 2153, and by 1.05 ± 0.93 nmol.g-1 fm from the youngest to the oldest internode in
line 2121. Sugarcane line 2153 contained 100 times more 1-kestose than 2121 in the
oldest sampled internode hence the lines were referred to as high- and low-1-kestose
producers. The production of 1-kestose did not reduce sucrose levels in the
transgenics, instead they contained significantly higher levels of sucrose than the
control line NCo310 (p<0.01, N=72). The production of this alternative sugar in addition
to elevated sucrose levels significantly increased the total sugar content in the
transgenic lines (p<0.01, N=72). Moreover, the high-1-kestose producer had
statistically more total sugar than the low-1-kestose producer (p<0.01, N=72).
Soluble acid invertase (SAI) and neutral invertase (NI, β-fructofuranosidase EC
3.2.1.26) from non-transgenic sugarcane internodal tissues were separated and
partially purified. Kinetic analysis of the purified invertases revealed two isoforms of SAI
eluting at approximately 100 mM KCl in a linear gradient while NI eluted at
approximately 500 mM KCl. The final specific activities of SAI and NI were 88.57
pkat.mg-1 protein and 92.31 pkat.mg-1 protein, respectively. This implied a 16- fold
purification of SAI, and 4- fold purification of NI. The pH optimum for NI was 7.0 and
that for soluble acid invertase less than 5.0. Due to the broad pH activities of the
invertases, activities significantly overlapped between pH 4.5 and 7.0. The affinity of
these invertases for 1-kestose hydrolysis was tested. The invertases displayed
hyperbolic saturation kinetics for sucrose, and had low affinities for 1-kestose with Km
values ranging from 50 - 247 mM. Furthermore, the presence of 200 mM 1-kestose had an inhibitory effect on SAI-mediated sucrose hydrolysis reducing activity to 51 % and
54 % for isoform 1 and 2 respectively.
To determine whether carbon allocation had been altered by the expression and
activity of 1-SST, 14C whole-plant radiolabelling experiments were conducted.
Radiolabelled CO2 was fed to the leaf subtending internode 5 and the allocation of
carbon to different parts of the culm was assessed. There was no significant difference
in the distribution of total radiolabel down the culm of the three sugarcane lines
(p>0.05, N=72). However, the percentage of total radiolabel in the water-soluble
fraction per internode in the high-1-kestose producer was significantly higher than the
other two lines (p<0.01, N=72). As a result, the percentage radiolabel in the waterinsoluble
fraction in this transgenic was concomitantly lower than in the other lines.
Carbon was therefore redirected from the water-insoluble fraction to the water-soluble
fraction to account for the additive production of 1-kestose. The expression of 1-SST in
sugarcane therefore established an additional carbohydrate sink by the flow of carbon
from the sucrose pool into 1-kestose. This did not lead to a depletion of the sucrose
pool, but rather stimulated carbon channelling into this pathway, thereby increasing the
non-structural carbohydrate content of the plant in one of the transgenics.
The work described in this study is the first to report on carbon partitioning in 1-
kestose-producing sugarcane grown under field resembling conditions. It contributes
significantly to an improved understanding of carbon partitioning in the culm, and
demonstrates that an alternative sugar can be produced in sugarcane under field
resembling conditions.
Description
Thesis (MSc (Genetics. Plant Biotechnology))--University of Stellenbosch, 2007.
Keywords
Theses -- Plant biotechnology, Dissertations -- Plant biotechnology