Carbon turnover and sucrose metabolism in the culm of transgenic sugarcane producing 1-Kestose

[摘要] Carbon partitioning was investigated in sugarcane (Saccharum spp. hybrids) that wasgenetically 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 fromone sucrose molecule to another to produce the trisaccharide 1-kestose. Molecularcharacterisation of four sugarcane lines, regenerated after transformation, confirmedthat two lines (2153 and 2121) were transgenic, with at least one intact copy of 1-SSTpresent in line 2153, and a minimum of five copies (or portions thereof) present in line2121. The novel gene was successfully transcribed and translated in both lines, asconfirmed by cDNA gel blot hybridisation and HPLC analysis respectively.Kestose production was stable under field resembling conditions and levels of thistrisaccharide 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 internode16 of 2153, and by 1.05 ± 0.93 nmol.g-1 fm from the youngest to the oldest internode inline 2121. Sugarcane line 2153 contained 100 times more 1-kestose than 2121 in theoldest sampled internode hence the lines were referred to as high- and low-1-kestoseproducers. The production of 1-kestose did not reduce sucrose levels in thetransgenics, instead they contained significantly higher levels of sucrose than thecontrol line NCo310 (p<0.01, N=72). The production of this alternative sugar in additionto elevated sucrose levels significantly increased the total sugar content in thetransgenic lines (p<0.01, N=72). Moreover, the high-1-kestose producer hadstatistically more total sugar than the low-1-kestose producer (p<0.01, N=72).Soluble acid invertase (SAI) and neutral invertase (NI, β-fructofuranosidase EC3.2.1.26) from non-transgenic sugarcane internodal tissues were separated andpartially purified. Kinetic analysis of the purified invertases revealed two isoforms of SAIeluting at approximately 100 mM KCl in a linear gradient while NI eluted atapproximately 500 mM KCl. The final specific activities of SAI and NI were 88.57pkat.mg-1 protein and 92.31 pkat.mg-1 protein, respectively. This implied a 16- foldpurification of SAI, and 4- fold purification of NI. The pH optimum for NI was 7.0 andthat for soluble acid invertase less than 5.0. Due to the broad pH activities of theinvertases, activities significantly overlapped between pH 4.5 and 7.0. The affinity ofthese invertases for 1-kestose hydrolysis was tested. The invertases displayedhyperbolic saturation kinetics for sucrose, and had low affinities for 1-kestose with Kmvalues 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 % and54 % for isoform 1 and 2 respectively.To determine whether carbon allocation had been altered by the expression andactivity of 1-SST, 14C whole-plant radiolabelling experiments were conducted.Radiolabelled CO2 was fed to the leaf subtending internode 5 and the allocation ofcarbon to different parts of the culm was assessed. There was no significant differencein 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-solublefraction per internode in the high-1-kestose producer was significantly higher than theother two lines (p<0.01, N=72). As a result, the percentage radiolabel in the waterinsolublefraction in this transgenic was concomitantly lower than in the other lines.Carbon was therefore redirected from the water-insoluble fraction to the water-solublefraction to account for the additive production of 1-kestose. The expression of 1-SST insugarcane therefore established an additional carbohydrate sink by the flow of carbonfrom the sucrose pool into 1-kestose. This did not lead to a depletion of the sucrosepool, but rather stimulated carbon channelling into this pathway, thereby increasing thenon-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 contributessignificantly to an improved understanding of carbon partitioning in the culm, anddemonstrates that an alternative sugar can be produced in sugarcane under fieldresembling conditions.

[发布机构] Stellenbosch University