German Title: Beeinflussung des Saccharoseverlustes nach der Ernte in Zuckerrüben : Evaluierung transgener Ansätze

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Abstract

Sugar beet (Beta vulgaris L.) is one of the economically most important plants storing high levels of sucrose. Sucrose is accumulated in the taproot inside the vacuoles of parenchyma cells, with up to nearly 20 % of fresh weight. Harvesting of sugar beet includes wounding of taproots, leading to induction of invertases and consequently to sucrose loss. In the present study, the interaction of the sugar beet invertase inhibitor BvC/VIF (Beta vulgaris cell wall and/or vacuolar inhibitor of ß-fructosidase) and its putative target enzymes was characterized and transgenic approaches, aiming at the modification of post-harvest sucrose metabolism investigated. Analysis of the inhibitor-invertase-interaction during wounding uncovered that complex formation between BvC/VIF and invertases does not necessarily lead to an inhibition of invertase activity. During the elucidation of prerequisites, needed for the down-regulation of invertase activity by proteinaceous inhibitors, site directed mutagenesis of BvVI1 (Beta vulgaris vacuolar invertase 1) demonstrated a putative involvement of the C-terminus of the invertase in the inhibition process. In order to intervene in post-harvest sucrose metabolism, sugar beet has been engineered to overexpress the endogenous invertase inhibitor BvC/VIF. Alternatively, the expression of endogenous BvC/VIF was silenced, with the purpose to determine the role of BvC/VIF within post-harvest sucrose-metabolism. The heterologous expression of BvC/VIF under control of the taproot specific 2-1-48 promoter did not lead to high expression levels, whereas under the control of the duplicated 35S-promoter, BvC/VIF was highly expressed and silenced via an RNAi construct, respectively. By overexpressing BvC/VIF, wound induced cell wall (CWI) as well as vacuolar (VI) invertase activity were strongly reduced. Notably, the extra-cellular localization of BvC/VIF was proven by a non-invasive approach and via immunolocalization, whereas no further evidence for an additional (earlier postulated) vacuolar localization was gained thus far. Unexpectedly, sucrose loss was not altered in BvC/VIF overexpressing lines, despite strongly reduced invertase activity. This observation led to the hypothesis of a demand-driven sucrose metabolism in wounded sugar beet taproots, based on altered activities of other sucrose hydrolytic enzymes in the case of hampered invertase activity. The determination of wound induced invertase activities in individual plants of BvC/VIF RNAi lines implicated an important role of BvC/VIF in regulating invertase activity after wounding, thus in limiting sucrose loss.

Translation of abstract (English)

Sugar beet (Beta vulgaris L.) is one of the economically most important plants storing high levels of sucrose. Sucrose is accumulated in the taproot inside the vacuoles of parenchyma cells, with up to nearly 20 % of fresh weight. Harvesting of sugar beet includes wounding of taproots, leading to induction of invertases and consequently to sucrose loss. In the present study, the interaction of the sugar beet invertase inhibitor BvC/VIF (Beta vulgaris cell wall and/or vacuolar inhibitor of ß-fructosidase) and its putative target enzymes was characterized and transgenic approaches, aiming at the modification of post-harvest sucrose metabolism investigated. Analysis of the inhibitor-invertase-interaction during wounding uncovered that complex formation between BvC/VIF and invertases does not necessarily lead to an inhibition of invertase activity. During the elucidation of prerequisites, needed for the down-regulation of invertase activity by proteinaceous inhibitors, site directed mutagenesis of BvVI1 (Beta vulgaris vacuolar invertase 1) demonstrated a putative involvement of the C-terminus of the invertase in the inhibition process. In order to intervene in post-harvest sucrose metabolism, sugar beet has been engineered to overexpress the endogenous invertase inhibitor BvC/VIF. Alternatively, the expression of endogenous BvC/VIF was silenced, with the purpose to determine the role of BvC/VIF within post-harvest sucrose-metabolism. The heterologous expression of BvC/VIF under control of the taproot specific 2-1-48 promoter did not lead to high expression levels, whereas under the control of the duplicated 35S-promoter, BvC/VIF was highly expressed and silenced via an RNAi construct, respectively. By overexpressing BvC/VIF, wound induced cell wall (CWI) as well as vacuolar (VI) invertase activity were strongly reduced. Notably, the extra-cellular localization of BvC/VIF was proven by a non-invasive approach and via immunolocalization, whereas no further evidence for an additional (earlier postulated) vacuolar localization was gained thus far. Unexpectedly, sucrose loss was not altered in BvC/VIF overexpressing lines, despite strongly reduced invertase activity. This observation led to the hypothesis of a demand-driven sucrose metabolism in wounded sugar beet taproots, based on altered activities of other sucrose hydrolytic enzymes in the case of hampered invertase activity. The determination of wound induced invertase activities in individual plants of BvC/VIF RNAi lines implicated an important role of BvC/VIF in regulating invertase activity after wounding, thus in limiting sucrose loss.