Characterisation of sucrose synthase activity in the sugarcane culm

[摘要] ENGLISH ABSTRACT: This study had three main goals:1. to investigate the occurrence on the protein level of sucrose synthase(SuSy) isoforms in sugarcane sink tissue,2. to determine the kinetic properties of these isoforms,3. to establish the tissue localisation of SuSy in the sugarcane culmThe results are summarised below:Three SuSy isoforms were obtained from leaf roll tissue. The SuSyA and SuSyBisoforms differed in terms of charge characteristics, with SuSyA not binding to ananion exchange column that bound SuSyB and SuSyC under the sameconditions. Both SuSyB and SuSyC isoforms were eluted at 180 mM KCl. TheSuSyA and SuSyB isoforms were present during autumn, but during winter onlythe SuSyC isoform could be isolated. Even though they eluted at the same saltconcentration, SuSyB and SuSyC were different isoforms, because they haddifferent kinetic parameters, as well as different immunological properties. SuSyBand SuSyC could not have been mixtures of the same isoforms, since apolyclonal antiserum against SuSyB, which inactivates native SuSyB, did notinactivate SuSyC. All three isoforms had significantly different kinetic parameters,with the SuSyA isoform also having a much lower sucrose breakdown/synthesisratio than the other two isoforms. Therefore, at least three SuSy isoforms occurin sugarcane leaf roll tissue on the protein level.The SuSyC isoform was subsequently kinetically characterised in detail. Datashowed that the enzyme employs an ordered ternary complex mechanism, withUDP binding first and UDP-glucose dissociating last. These experimentallyobtained kinetic parameters were then used to extend a kinetic model of sucroseaccumulation. Data show that when the experimentally determined SuSy kineticparameters were entered into the model, a 40 % increase in sucroseconcentration and 7 times reduction in fructose concentration resulted. Thesedata illustrate the pronounced physiological effects that may result from thepresence of different SuSy isoforms.SuSy protein localisation data, obtained by an immunohistochemical approach,indicated that SuSy protein was present in both storage parenchyma andvascular tissue of young, intermediate, and mature internodes. SuSy enzymeactivity in different parts of the internodes was similar, except for internode 3,which had much higher activity in the bottom part of the internode, possiblybecause growth is faster here, hence a higher demand for sucrose cleavageexists here.

[发布机构] Stellenbosch University