In planta modification of the potato tuber cell wall

Apart from its well known uses in the human diet a large amount of the grown potatoes (about one third in the Netherlands) is used for the isolation of starch which is used in several food and non-food applications. The cell wall fibres comprise a large portion of the waste material remaining after the starch isolation process. While cell wall fibres from some other plant species are used in food and non-food industry, a structural alteration of the potato fibres is necessary before similar applications are possible. However, before it is possible to generate plants with a tailor-made cell wall composition, questions concerning the best approach have to be answered and the necessary tools will have to be identified and made available. This thesis describes the results of a study investigating the possibilities to generate transgenically modified potato plants with an altered cell wall composition. These experiments were mostly focussed on altering pectin composition. This is particularly interesting because several studies already showed that many different pectin structures occur in specific plants, plant tissues and developmental stages. Plants with a specific alteration in pectin structure may aid in revealing the biological significance of these different structures. Additionally, the possibility to produce a particular pectin structure may be useful for the food industry, in which pectins from other plant species are already used as a gelling agent. At the start of the work described in this thesis only few genes involved in cell wall biosynthesis were identified which favoured the heterologous expression of fungal pectin degrading enzymes. A rhamnogalacturonan lyase ( e RGL) from Aspergillus aculeatus , which is able to cleave the rhamnogalacturonan I (RG I) at specific sites, was introduced. The e RGL was successfully expressed (under control of the granule-bound starch synthase promoter) and translated into an active protein, demonstrated by e RGL activity in the tuber extracts. These tubers showed clear morphological alterations, including radial swelling of the periderm cells and development of intercellular spaces in the cortex. Sugar compositional analysis and antibody labelling studies showed a large reduction in galactan and arabinan side-chains of RG I. These data show the possibility of specifically modifying cell wall polysaccharide structures by the introduction of such a pectin degrading enzyme. Additionally, the results suggest that RG I has an important role in anchoring galactans and arabinans at particular regions in the wall and in normal development of the periderm. The utility of these transgenic plants in answering questions concerning the biological importance of cell wall polysaccharides is evident. Apart from modifying the cell wall composition by the introduction of pectin degrading enzymes two experiments were performed focussed on interference with the biosynthetic machinery of the plant cell wall at different levels. The first study concerns the modulation of cellulose synthase ( CesA ) gene expression. Since this enzyme is polymerising theb-1,4 glucan chains forming cellulose, its altered expression is likely to directly affect the level of cellulose in the wall. In the second study the expression of the UDP-Glc-4-epimerase ( UGE ) was modulated. The UGE is responsible for the conversion of UDP-glucose to UDP-galactose and vice-versa. Its altered expression is likely to affect the amount of cell wall bound galactan. Four CesA genes were isolated from potato and one full length cDNA clone was used for up- and down-regulation of the corresponding RNA expression levels controlled by the granule-bound starch synthase promoter. Fourier Transform Infra-Red microspectroscopy (FTIR) was used for the identification of transformants with altered levels of cellulose in their tuber cell walls in comparison to WT plants. A further quantification of these results, by measuring the cellulose content in the cell wall material, showed that by modulating the CesA expression levels, tubers with levels of cellulose ranging from 50 to 200% of the WT amount were obtained. Especially the increase in cellulose is quite remarkable and in contradiction with the general believe that expression of more than one CesA gene (and possibly even more genes) is necessary to achieve such a modification. By using a specific region of the other three CesA genes in antisense experiments we managed to individually down regulate these genes and concomitantly the cellulose levels in the tubers of these plants. The use of this so-called class specific region (CSR), which is only present in plant cellulose synthases and is believed to determine the genetic difference between the different CesA genes in one plant, showed to be sufficient to down regulate the corresponding gene. In contrast to many other plants and plant systems, depletion of cellulose (to 50% of WT level) in potato tubers did not result in any phenotypic alterations. However, our potato plants were grown at normal conditions while some of the cellulose synthase mutants only revealed a phenotype when grown at restrictive conditions. Another important result is the fact that not all potato transformants with decreased cellulose levels show modifications in their pectin composition. This indicates a delicate balance between cellulose and pectin levels and that an altered pectin composition in plants with depleted cellulose is not necessarily a response upon reduced strength of the cell wall. For the UDP-Glc-4-epimerase two potato cDNA clones ( StUGE45 and StUGE51 ) were identified and used for overexpression in potato tubers. The increased levels of cell wall bound galactan in these tubers indicates the importance of UDP-galactose levels for galactan deposition in the cell wall. Additionally these plants showed a small decrease in the amount of galacturonan. This suggests that alterations in the UDP-galactose pool size can influence the levels of nucleotide sugars which are used for the synthesis of other polysaccharides. Additionally, the elevated expression levels of the two UGE s showed to have different effects, which suggests that they have a different function in plant development. Further research has to show whether other polysaccharides than cell wall galactan are affected by this decrease in galactose. Xyloglucan and galactomannan also contain galactosyl residues and the decrease does not necessarily affect the levels of RG I bound galactan. Both these studies show the possibility to induce alterations in the cell wall composition by interfering with the biosynthetic machinery. Identification of more genes involved in cell wall biosynthesis is necessary to enable new studies in the future. An RNA fingerprinting experiment was performed to investigate the possibility of identifying new genes involved in primary cell wall biosynthesis. Potato leaf protoplasts showed to regenerate a new cell wall in the first 18h after transfer to a culture medium. At 5 distinct time-points RNA was isolated and the expressed genes were visualised using cDNA-AFLP. Around 8500 transcript derived fragments (TDFs) were visualised from which 156 were isolated and sequenced. However, no cell wall related TDFs were identified. This indicates that even though the protoplasts actively regenerate a new cell wall, this did not result in highly increased expression of genes involved in cell wall biosynthesis or modification. In summary the experiments described in this thesis showed that different approaches can be used to generate a modified cell wall composition in potato tubers. These genetically modified plants have shown to be an interesting study material for unravelling the biological function of different cell wall polysaccharide structures. Additionally these transformants obviously showed that the potato tuber cell wall is amenable to genetic modification. There is a wide range of wall modifications which is tolerated by the tubers, which may hold a promise for the future in valorising the fibre fraction of potato after starch isolation.