Fructan biosynthesis in crop plants : the molecular regulation of fructan biosynthesis in chicory (Cichorium intybus L.)

Abstract

Fructan is a polymer of fructose produced by plants and microorganisms. Within the plant kingdom about 45.000 species accumulate fructan as storage carbohydrate in addition to, or instead of, starch. Fructan accumulating species are mainly found in temperate and sub-tropical regions with seasonal or sporadic rainfall. During the last decades, the use of fructan in the (food) industry has rapidly evolved, because of its health promoting characteristics and interesting functional properties.Chicory (Cichorium intybus L.) is a biennial taproot-bearing crop plant that is grown for the production of inulin on an industrial scale. Inulin, a ß(2,1) linked linear fructan with a terminal glucose residue, is stored in the chicory taproots. The degree of polymerisation (DP) determines the application of the inulin and hence the value of the crop. This leads us to the central question of this thesis: What regulates the fructan yield and the degree of polymerisation, and how can we modify this? The DP is highly dependent on the field conditions and harvest time, and therefore the first step in answering this question was tostudy the regulation of fructan (inulin) metabolism throughout the growing season. This is described in Chapter 2. Metabolic aspects of inulin production and degradation in chicory were monitored in the field and under controlled conditions. We determined the concentrations of soluble carbohydrates, the inulin mean degree of polymerisation (mDP), inulin yield, gene expression and activity of enzymes involved in inulin metabolism in the taproots. Inulin biosynthesis - catalysed by sucrose: sucrose 1-fructosyltransferase (EC 2.4.1.99) (1-SST) and fructan: fructan 1-fructosyltransferase (EC 2.4.1.100) (1-FFT) - started at the onset of taproot development. Inulin yield increased with time following a sigmoid curve reaching a maximum in November. The maximum inulin mDP of 15 was reached in September and then gradually decreased. Based on the changes observed in the pattern of inulin accumulation, we defined three phases in the growing season and analysed product formation, enzyme activity and gene expression in these defined periods. The results were validated by performing experiments under controlled conditions in climate rooms. Our results show that the decrease in 1-SST is not regulated by day length and temperature. From mid-September onwards the mDP decreased gradually although inulin yield still increased. This is most probably the result from back-transfer activity of 1-FFT and fructan exohydrolase activity (EC 3.2.1.153) (1-FEH). In plants 1-FEH catalyses the breakdown of fructan in order to release the stored carbohydrates necessary in periods of stress, like cold or drought periodsor flowering. This information was used to design two strategies to obtain the desired, increased inulin DP and yield. Overexpression of 1-SSTwas performed to increase the mDP and to keep the sucrose concentration low, to prevent 1-FFT from depolymerizing inulin. The result was a higher mDP during the growing season. Unfortunately, no effect on the mDP was seen at the end of the growing season, most probably due to activity of FEH. Secondly, anFEH I antisense fragment was introduced into chicory in order to block depolymerisation at the end of the growing season. This resulted in a reduction in FEH Iexpression upon cold induction, but had only minor effects on the mDP. The degradation of inulin was most probably caused by the remaining 1-FEH activity. Overall this study showed that inulin metabolism in chicory is tightly regulated, but also revealed options to further steer inulin metabolism in chicory. The next step in answering the central question was to study the regulation of the genes involved in fructan biosynthesis. In Chapter 3this was studied at three different levels. Firstly, fructan gene expression and carbohydrate concentrations were studied in axial sections of mature chicory root, revealing the highest expression levels and carbohydrate levels in the phloem. Correlations were found between the gene expression patterns of 1-SST, 1-FFT and the carbohydrate levels, suggesting a possible involvement of sugars in the regulation of 1-SSTand 1-FFTgene expression. Secondly, the induction of 1-SSTand 1-FFTexpression was studied in excised chicory leaves. Expression of both 1-SSTand 1-FFTwas induced upon sucrose and glucose feeding, suggesting that both genes are at least partly regulated in the same way. Upon fructose feeding, the induction of fructan biosynthesis was less pronounced than with sucrose. The expression of 1-SSTwas induced by fructose but this resulted in only low amounts of 1-kestose. The expression of 1-FFTwas not induced upon fructose feeding.Thirdly, to further unravel the mechanism of induction, the promoters of 1-SSTand 1-FFTfrom chicory were isolated and characterized through in silicoand in planta(only 1-FFT) analysis. Computational analysis of fructosyltransferase (FT) promoters revealed elements that are common in fructan biosynthesis-promoters among different species and also occur in Arabidopsis promoter sequences. One of these elements is predominantly present in genes involved in sugar metabolism and transport. This element did also contain a core sequence involved in MYB transcription factor binding important for fructan biosynthesis activation in wheat, as was published recently. An 1100bp 1-FFTpromoter fragment was shown to be functional in transgenic chicory and in the non-fructan accumulating plants species, Arabidopsis and potato. Application of carbohydrates resulted in the expression of the reporter gene GUS comparable to 1-FFTexpression upon carbohydrate feeding in chicory. This study provides information on the regulation of inulin biosynthesis, suggestions for studies on transcription factors, and provides a promoter for steering the expression of fructan biosynthetic genes in transgenic plants. An alternative way for the production of inulin with the desired DP and yield, circumventing the problems in chicory rather than trying to solve them, is the introduction of the fructan biosynthetic pathway in non-fructan metabolizing and catabolizing plant species. Towards this end we have expressed the inulin synthesizing enzymes, 1-SST and 1-FFT from Jerusalem artichoke, in maize and potato, as described inChapter 4. Transgenic maize plants produced inulin type fructan (at 3.2 milligram per gram kernel) and kernel development was not affected. Potato tubers expressing 1-SSTaccumulated 1.8 milligram inulin per gram tuber while tubers with a combined expression of 1-SSTand 1-FFTaccumulated 2.6 milligram inulin per gram tuber. Inulin accumulation in maize kernels was modulated by kernel development, first peaking in young seeds and then decreasing again through degradation during late kernel development. In potato, inulin mDP was relatively stable throughout tuber development and little evidence of degradation was observed. The accumulation of 1-kestose in transgenic maize correlated positively with kernel sucrose concentration and introduction of the fructan biosynthetic pathway in a high-sucrose maize background increased inulin accumulation to 41 milligram per gram kernel kernel. This study shows the importance of sugar availability and the absence of degradation mechanisms in platform crops for tailor-made fructan production. Further evaluation of the production of tailor-made inulin and putative platform crops is discussed in Chapter5.Here we come to the conclusion that the mDP, the distribution and yield depend on the origin of the fructan biosynthesis genes and the availability of sucrose in the host. The combination of genes from different origins could result in new types and different lengths of fructan molecules resulting in (new) specific properties of fructan. Limitations for the production of tailor-made fructan in chicory are not seen in putative new platform crops, such as sugar beet, sugarcane and rice. The work described in this thesis on fructan biosynthesis in chicory and in new platform crops has resulted in new insights that will lead new applied and fundamental research in this field.</p